POPULATION GENETIC STRUCTURE AND TAXONOMIC EVALUATION OF
TWO CLOSELY RELATED FRESHWATER MUSSEL SPECIES, THE EASTERN
FLOATER, PYGANODON CATARACTA, AND THE NEWFOUNDLAND FLOATER,
P. FRAGILIS, IN ATLANTIC CANADA
by
LJILJANA MARIJA STANTON
Thesis Submitted in partial fulfillment of the requirements for The Degree of Master of Science (Biology)
Acadia University Fall Convocation 2008
© by LJILJANA MARIJA STANTON, 2008 I, L. M. Stanton, grant permission to the University Librarian at Acadia University to reproduce, loan, or distrubute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis.
Signature of Author
Date This thesis by Ljiljana Marija Stanton was defended successfully in an oral examination on September 2, 2008.
The examining committee for the thesis was:
Dr. John Murimboh, Chair
Dr. W.R. Hoeh, External Reader
Dr. Stephen Mockford, Internal Reader
Dr. D.T. Stewart, Supervisor
Dr. M. Synder, Head
This thesis is accepted in its present form by the Division of Research and Graduate Studies as satisfying the thesis requirements for the degree Master of Science (Biology). TABLE OF CONTENTS
List of tables vi
List of figures vii
Abstract ix
Acknowledgements x
Chapter 1. Natural history of freshwater mussels: implications for conservation 1
Life history traits of the Family Unionidae 1
Taxonomy and natural history of Pyganodon cataracta and P. fragilis 4
Molecular markers 14
Chapter 2. Population structure and taxonomic status of Pyganodon cataracta and P. fragilis inferred from AFLP and sequence data 18
Introduction 18
Methods 22 Sample collection and DNA isolation 22 Morphology analysis 29 DNA sequencing and data analysis 30 AFLP procedure and data analysis 34
Results 39 Morphological analysis 39 Mitochondrial and nuclear DNA sequencing analysis 42 AFLP analysis 49
Discussion 54 Taxonomic implications 54 (A) Morphology 54 (B) Genetic divergence 56 (C) Population genetic structure 62 Historical biogeography 65 Conservation implications 67
iv Chapter 3. General conclusions and recommendations for future conservation of freshwater mussels 71
Literature cited 76
v List of Tables
Table 2.1. Description of sampling localities by province, subdrainage
area and site location of both species collected, including sample
size for the COI, ITS-1 and AFLP analysis 27
Table 2.2. List of reference specimens and outgroup species used in the
COI and ITS-1 analysis, their locality and GenBank accession
numbers
Table 2.3. Selected primer sequences and primer combinations used
in the AFLP analysis.
Table 2.4. Genetic distances (Kimura two parameter model) of cytochrome C
oxidase I sequences between outgroup species Strophitus undulatus,
P. grandis, P. lacustris and P. cataracta and P. fragilis. List of Figures
Figure 1.1. Suspected geographic distribution of P. cataracta and P. fragilis
in Eastern Canada. 8
Figure 1.2. Illustration of the conchological features of P. cataracta and
P. fragilis. 10
Figure 2.1. Map of Pyganodon sampling locations in Atlantic Canada. 26
Figure 2.2. Principle component analysis biplot showing the relationship
between P. cataracta and P. fragilis individuals based on common
morphological measurements. 40
Figure 2.3. UPGMA dendogram based on common morphological
characteristics of the presumptive "P. cataracta" and"P. fragilis"
specimens from Nova Scotia and New Brunswick. 41
Figure 2.4. Parsimony network of P. fragilis and P. cataracta mtDNA
haplotypes for the COI gene. 43
Figure 2.5. 50% majority rule consensus tree (rooted) produced by a neighbor-
joining analysis of the cytochrome c oxidase subunit 1 (COI)
sequence data from 128 Pyganodon specimens. 45
Figure 2.6. 50% majority rule consensus tree (unrooted) produced by neighbor
joining and maximum parsimony analyses of the cytochrome c oxidase
subunit 1 (COI) sequence data from 128 Pyganodon specimens. 46
vn Figure 2.7. 50% majority rule consensus tree (rooted) produced by a maximum
parsimony analysis of the cytochrome c oxidase subunit 1 (COI)
sequence data from 128 Pyganodon specimens. 47
Figure 2.8. 50% majority rule consensus tree (rooted) produced by a maximum
likelihood analysis of the cytochrome c oxidase subunit 1 (COI)
sequence data from 128 Pyganodon specimens. 48
Figure 2.9. 50% majority rule consensus tree (rooted) produced by a both
neighbor-joining and maximum parsimony analysis of the internal
transcribed spacer region (ITS-1) sequence data from 20 Pyganodon
specimens. 50
Figure 2.10. Neighbor-joining analysis of 35 Pyganodon specimens based on
data from 235 AFLP loci with 100 bootstrap replicates. 51
Figure 2.11. Graphs depicting the methods used for determining value of K or
number of groups from the AFLP data set. 52
Figure 2.12. A triangle plot displaying the estimated membership coefficient
for each individual into its assigned population (Newfoundland, New
Brunswick or Nova Scotia). 53
viii Abstract
The freshwater mussels Pyganodon cataracta and Pyganodonfragilis are both found in Atlantic Canada but their taxonomic status and exact geographic distribution is uncertain. Phenotypic plasticity and convergence of shell characteristics has created difficulties in delineating unionids based on morphological traits. Our study will utilize the mitochondrial cytochrome c oxidase (COI) gene, the nuclear internal transcribed spacer region (ITS-1) gene and a multi locus marker, amplified fragment length polymorphisms (AFLPs), in conjunction with morphological data to determine if
P.cataracta and P. fragilis are two distinct species, just one species, or a mixed hybridizing population. Mussels collected from Newfoundland, Nova Scotia, New
Brunswick and Prince Edward Island displayed low levels of genetic variability and extremely low divergence values for both the COI and ITS-1 sequence data, forming a single monophyletic group. Population genetic structure inferred from 235 AFLP loci, showed a moderate level of geographic structuring based on province, however no genetic differentiation among the putative species was observed. Common morphological characteristics used to distinguish the species in question were found to be unreliable. Our holistic approach of combining morphology, mitochondrial and nuclear data clearly indicates that P. cataracta and P. fragilis are not distinct species and should be regarded as a single species, with P. cataracta having nomenclatural priority. These data also have implications for understanding the historical biogeography of P. cataracta and for conservation strategies for this species in eastern Canada.
ix Acknowledgements
I can not begin to explain how rewarding and enriching this experience has been for me. I would like to thank, first and foremost, my supervisor Don Stewart who provided me with this opportunity and has guided me along the way, together with all the wonderful people in the Biology Department at Acadia who have allowed me to learn and grow as a biologist. Specifically at Acadia, I would like to thank Rodger Evans for supplying the AFLP reagents, Steve Mockford for all the insightful discussions and
Nancy and Wanda for always being so kind and helpful. A special thanks to Aaron
Shafer and Mamta Jha in the DTS lab for all your advice and assistance and to my fellow graduate students and friends for our memorable gatherings- I've enjoyed getting to know you all through the past few years.
None of this work would have been possible without the hard work and dedication of fellow mussel enthusiasts Randy Hoeh, John Maunder and Don McApline, who collected and donated Pyganodon samples from across Atlantic Canada. Financial support for this research was primarily funded by the New Brunswick Wildlife Trust
Fund, in addition to, the Acadia University Research Fund and the National Science and
Engineering Research Council of Canada (via Don Stewart). Acadia Graduate Awards and the Dr. Robert Graeme Boutilier Memorial Research Award provided financial assistance for my stipend.
x Finally, I would like to thank my family Barret, Renee and especially my Mom for their continued support and enthusiasm for whatever it is I choose to do. To my father for unwittingly instilling in me a love of science, this thesis is dedicated to your lasting memory. Last but certainly not least my wonderful, loving husband you are definitely the best. I cannot thank you enough for supporting me every step of the way, providing advice and encouragement, a listening ear, and a helping hand in collecting mussels - you truly deserve a honourary degree.
'The essential purpose is to decide for oneself what is of genuine value in life. And then to find the courage to take your own thoughts seriously' - Albert Einstein.
xi Chapter 1. Natural history of freshwater mussels: Implications for
Conservation
Life history traits of the Family Unionidae
Freshwater mussels (Order Unionoida) are a fascinating yet widely overlooked group of animals whose populations are drastically declining in many areas and are considered among the most endangered groups of organisms in North America (Williams et al, 1993; Lydeard et al, 2004). Freshwater mussels are broadly distributed on all continents except Antarctica but they attain the greatest diversity in North America where over 300 species have been described (Williams et al, 1993; Turgeon et al, 1998, Roe and Hoeh, 2003). Despite their relatively high diversity, the Phylum Mollusca has the greatest number of recorded extinctions of any taxonomic group (Lydeard et al, 2004) and of the freshwater mussels in North America, 70% of species are considered endangered, threatened or are of special concern (Williams et al, 1993). A combination of life history traits and anthropogenic effects such as increased pollution, sedimentation, habitat destruction, excessive damming of rivers as well as the introduction of exotic species such as the Asiatic clam, Corbiculafluminea, the zebra mussel, Dreissena polymorpha and the quagga mussel, D. burgensi, has all contributed to their decline
(Williams et al, 1993; Bogan, 1993; Lydeard et al, 2004).
1 Extant freshwater mussels have a fossil record dating back to the early Triassic
(Haas, 1969; Good, 1998), with all modern families present by the end of the Cretaceous
(Watters, 2001). Bivalves of the order Unionoida, commonly known as unionoids, are divided into two superfamilies the Unionoidea (containing three families: Hyriidae,
Margaritiferidae, and Unionidae) and the Etherioidea (containing three families:
Etheriidae, Iridinidae, and the Mycetopodidae) based on larval morphology (Roe and
Hoeh, 2003). The Unionoidea have unique bivalved larvae referred to as glochidia
(singular glochidium) and are broadly distributed across North America, Eurasia,
Australasia, South America and Africa, whereas the Etherioidea possess a univalved larva referred to as lasidia or haustoria and are restricted to Central and South America,
Africa and India (Roe and Hoeh, 2003). The larval types of the unionoidean and etheroidean bivalves are so distinct morphologically it was hypothesized by Parodiz and
Bonetto (1963) that unionid bivalves may represent a polyphyletic assemblage and that these two lineages may have independently invaded freshwater. However, recent phylogenetic and molecular analyses have rejected this claim and support the hypothesis that the unionoids are a monophyletic group (Hoeh et al., 2001; Roe and Hoeh, 2003;
Walker et al., 2006).
Typically, members of the order Unionoida possess a unique life history trait; an obligate parasitic larval stage during which they depend upon the availability of a fish host to complete their reproductive cycle (Neves et al, 1985). Female unionids will brood the glochidia in a modified portion of their gills called the marsupium and will release their larvae when they detect a passing fish (Clarke, 1981). Some
2 species produce packages of glochidia called conglutinates (Kat, 1983). Other species modify aggregate their conglutinates to produce superglutinates that mimic fish prey or small fish that they use to lure their hosts (Haag et al., 1995). Once a suitable host is found, the larvae will attach to the gills or fins of fish, develop into juvenile mussels, and eventually release themselves to begin a free-living, benthic lifestyle. Mussels are considered to be either host generalists - parasitizing a wide variety of fish (Trdan and
Hoeh, 1982), or are host specific - parasitizing one to a few closely related fish species
(Zale and Neves, 1982; Yeager and Saylor, 1995). The decline or loss of natural fish populations may, therefore, adversely affect freshwater mussel populations by limiting their dispersal and reproductive success. Furthermore, conservation efforts are inhibited because fish hosts of most freshwater mussels have not yet been confirmed (King et al.,
1999). The knowledge offish hosts plays a critical role in developing effective conservation programs (Haag and Warren, 1997) as well as providing important information on the abundance and distribution of mussels (Watters, 1992).
Unionids are able to tolerate a wide variety of aquatic environments, and inhabit both lotic and lentic habitats (Trdan and Hoeh, 1982). Factors that affect freshwater mussel distributions include: sediment type, and depth, velocity, pH, and temperature of water. Freshwater mussels are most successful at medium water velocities thus avoiding the effects of siltation at lower water velocities and the absence of substrate stability at higher water velocities (Salmon and Green, 1982).
3 The presence and diversity of freshwater mussels are thought to be important indicators of a healthy aquatic ecosystem because their presence reflects good water quality and habitat characteristics (Williams et al., 1993). As the dominant filter feeders in many lakes and rivers, unionids are considered to be 'keystone taxa' directly affecting nutrient dynamics and benthic processes, as well as creating habitats for other organisms that may live on or around their shells (Maclsaac, 1996; Vaughn and Hakenkamp, 2001;
Gutierrez et al., 2003). A recent study suggested that mussel abundance could be used as an effective bioindicator or as indicator taxa of general freshwater biodiversity in lowland rivers in the UK, where a high abundance of mussels supported a higher proportion of other invertebrate taxa (Aldridge et al., 2007). This approach of using mussels as indicator taxa would be useful in other regions of the world such as North America to access the biodiversity of freshwater ecosystems in addition to providing a survey of current mussel distributions and areas of conservation priority.
Taxonomy and Natural History of Pyganodon cataracta and P.fragilis
Of the approximately 300 freshwater mussels species found in North America, 55 have been recorded in Canada (Williams et al., 1993; Metcalfe-Smith et al., 1998) and only 12 are found in Atlantic Canada. Several of these are of conservation concern. The
Dwarf wedgemussel, Alasmidonta heterodon, once found in New Brunswick is now considered extirpated in Canada (Hanson and Locke, 2000). The Yellow lamp mussel,
Lampsilis cariosa, occurs only in the St. John River and in the Sydney River watersheds
4 and is listed as a species of special concern in both New Brunswick and Nova Scotia by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC). Although
COSEWIC has recognized the vulnerability of freshwater mussels in Canada, the remaining nine species found in Atlantic Canada have not been directly assessed by
COSEWIC. An assessment of freshwater mussels was conducted by Metcalfe-Smith and
Cudmore-Vokey for Environment Canada in 2004 for all species in Canada and this document is awaiting official review by COSEWIC. Only 19 of the 55 Canadian species
(35%) were ranked as nationally secure, highlighting the urgent need for conservation studies of unionids in Canada (Metcalfe-Smith and Cudmore-Vokey, 2004).
Both Pyganodon cataracta, the Eastern floater, and Pyganodonfragilis, the
Newfoundland floater, are thought to be relatively common in parts of their range but their taxonomic status and exact geographic distribution within Atlantic Canada remain unclear. Indeed, the province of New Brunswick has reserved assigning conservation status to these species because of their taxonomic uncertainty. Species identification within the unionids is frequently complicated by phenotypic plasticity and convergent evolution of shell characteristics (Roe and Hoeh, 2003). The taxonomy of P. cataracta and P. fragilis is no exception. Historically, these two putative species have caused considerable debate among taxonomists, due to subtle morphological differences that exist between them and indeed some taxonomists have suggested that the "diagnostic" characteristics of these species such as shell colouration and stomach anatomy are unreliable (Hanlon and Smith, 1999).
5 A taxonomic revision of Eastern North American Anodonta, based on a study conducted by Hoeh (1990), utilizing both allozyme and morphological data resulted in the subgenus Pyganodon being raised to distinct genus level due to extremely high genetic divergence from the subgenus Anodonta. Originally, Pyganodon (=Anodonta) cataracta and Pyganodon (=Anodonta) fragilis were described and named as separate species by Say, 1817 and Lamarck, 1819, respectively. The type specimens for these two species were thought to be collected in Pennsylvania and Newfoundland, respectively.
The genus Pyganodon consists of five North American species including P. cataracta, P. fragilis, P. grandis, P. gibbosa and P. lacustris. P. gibbosa is endemic to one river system in the state of Georgia. P. lacustris occurs in the St. Lawrence, upper
Mississippi and southern Hudson Bay drainages (Hoeh and Burch, 1989). P. grandis is common and widespread across Canada and the United States and is considered abundant and generally secure across its entire range. Many subspecies of P. grandis (i.e. P. grandis corpulenta (Cooper, 1834), P. grandis simpsonia (Lea, 1861) and P. grandis grandis (Say, 1829)) have been proposed but other authors (e.g., Cummings and Mayer,
1992; Park and Burch, 1995; Metcalfe-Smith and Cudmore-Vokey, 2004) are not convinced that these represent distinct morphological forms of P. grandis. In addition, despite P. lacustris being described as a distinct species based on conchological and allozyme evidence provided by Hoeh and Burch (1989), it is still considered by some to be yet another morphological variant of P. grandis (Strayer and Jirka, 1997).
6 According to The Freshwater Mussels of Canada (Clarke, 1981), Pyganodon cataracta is widely distributed throughout most of Eastern North America, ranging from the lower St. Lawrence River drainage, to the Atlantic provinces and as far south as the
Gulf of Mexico drainage (Figure 1.1a). Metcalfe-Smith and Cudmore-Vokey (2004), report P. cataracta as abundant and secure throughout its range in Nova Scotia, New
Brunswick, Prince Edward Island and Quebec; however, the species might be at risk within Ontario where only one peripheral population has been identified in the Ottawa
River.
Pyganodon fragilis \s likely a Canadian endemic species (i.e., if it occurs only in
Atlantic provinces, Ontario and Quebec) but its status could not be determined due to no confirmed records or specimens from either New Brunswick or Nova Scotia, therefore the status of this species were not assessed by Metcalfe-Smith and Cudmore-Vokey
(2004). Populations in Quebec were listed as sensitive whereas populations in
Newfoundland were undetermined due to taxonomic uncertainty and the unconfirmed possibility of two or more species in Newfoundland and Labrador (John Maunder, pers. comm).
According to Clarke (1981), "typical" P. fragilis specimens are thought to occur primarily in Newfoundland, however, specimens displaying intermediate characteristics between P. fragilis and P. cataracta are commonly found in Nova Scotia, New
Brunswick and eastern Quebec (Figure 1.1b). Historical data suggested that the range of
P. fragilis may have extended into the northeastern United States however, a study
7 examining the conchology and stomach anatomy of mussels collected in Maine suggested that those specimens were actually P. cataracta that were misidentified as P. fragilis
(Hanlon and Smith, 1999).
Figure 1.1. Suspected geographic distribution of a) P. cataracta and b) P. fragilis in Eastern Canada. The map has been modified from Clarke, (1981).
First records of Pyganodon cataracta in Nova Scotia were apparently identified by J.R. Willis in 1863 as Anodonta fernssaciana and many early taxonomists described morphological variants of P. fragilis as different species. For example, Anodonta marginata was described by Say in 1817 and Anodonta brooksiana was identified as a distinct population in Newfoundland by Van der Schalie in 1938. Following a reexamination of these specimens in 1963, Clarke and Rick concluded that
8 both Anodonta marginata and Anodonta brooksiana are synonymous and cannot be distinguished from the original descriptions of P. fragilis made by Lamarck. Based on the presence of intergrades between P. cataracta and P. fragilis, Clarke and Rick (1963) stated that although these two putative species can be distinguished morphologically, they are not reproductively isolated and therefore they, recommended that P. fragilis should be considered a subspecies of P. cataracta. There has been some speculation as to whether or not these taxa are undergoing hybridization in part of their range and biologists continue to debate their genetic distinctiveness (Hanlon and Smith, 1999). Support for recognizing P. fragilis as a unique species came from some preliminary genetic studies conducted by Hoeh (1990) who suggested that P. fragilis may only occur in
Newfoundland and Labrador, making it unlikely that hybridization is occurring between the two taxa, as their ranges may not overlap.
As described by Clarke (1981), P. cataracta is generally the larger of the two taxa. Its shell dimensions may reach up to 150mm in length, 75mm in height and 65mm in width as opposed to P. fragilis, which may reach up to 90mm in length, 45mm in height, and 25mm in width. Both are elliptical in shape and possess thin, fragile shells with P. cataracta exhibiting a more rounded posterior end and P. fragilis a more blunt posterior end (Figure 1.2a).
The periostracum or outer shell of P. cataracta is described by Johnson (1970) as having a smooth, shiny surface with relatively few growth rests. The shell is commonly grass-green with yellow or brown tones covered in narrow or broad green rays
9 while some are completely brown in colour. P. fragilis has either a "shiny or dull, straw yellow to brown periostracum" with prominent growth rests and obscure greens ray
(Johnson, 1970). The nacre or inner shell of both species is silvery white and tinted with blue or yellow. However, most P. fragilis specimens have yellow blotches or spots and are centrally discoloured (Figure 1.2b) (Clarke, 1981). One major characteristic that has been used to differentiate these species is the presence of a double loop beak structure
(curved concentric bars) on the umbone of P. cataracta and a single loop or irregular folds seen on the umbone of P. fragilis (Clarke, 1981) (Figure 1.2c). Unfortunately, this loop structure is often destroyed by erosion of the umbone particularly in larger/older individuals (Figure 1.2d).
Figure 1.2. Illustration of the conchological features of P. cataracta and P. fragilis detailing the (a) posterior end of the shell (b) nacre (c) umbone and (d) erosion of umbone. This figure has been modified from Clarke, (1981).
10 Although not many studies have detailed the reproductive biology off. cataracta and P. fragilis, females are thought to be winter brooders in Atlantic Canada (Threlfal,
1986), releasing their glochidia onto a suitable fish host in either April or May (Clarke,
1981). The glochidia of both species are similar in appearance. Both are hooked and triangular in shape, however, the glochidia of P. cataracta were found to be significantly smaller than those of P. fragilis (Wiles, 1975). Species of Anodonta and possibly
Pyganodon are thought to be eurytopic with respect to host use (i.e., they may parasitize a wide variety offish species - Trdan and Hoeh, 1982). Confirmed fish hosts of P. cataracta from Atlantic Canada are the Threespine Stickleback, Gasterosteus aculeatus, from Newfoundland (Threlfall, 1986) and the White Sucker, Castostomus commersoni, the Pumpkinseed Sunfish, Lepomis gibbosus, and the Yellow Perch, Percaflavescens, from Nova Scotia (Wiles, 1975). A recent study in New Brunswick uncovered a large number of potential fish hosts including the Common Shiner, Luxilus cornutus, the
Blacknose Dace, Rhinichthys atratulus, Creek Chub, Semotilus atromaculatus, and the
Ninespine Stickleback, Pungitius pungitius (Beaudet, 2006). Other reported fish hosts of
P. cataracta from its southern American range include the rock bass, Ambloplites rupestris, the common carp, Cyprinus carpio, and the bluegill, Lepomis macrochirus
(Strayer and Jirka, 1997; Nedeau et ai, 2000; Bogan, 2002). Although no host fish for P. fragilis have been verified, Nedeau et al. (2000) believe they may use the same or similar fish hosts as P. cataracta.
Pyganodon cataracta and P. fragilis appear to occupy similar habitat types and are found in ponds, lakes, streams, river ox-bow ponds and beaver ponds. Both species
11 reportedly prefer muddy or silty substrates but can also been found in sand or occasionally gravel (Clarke, 1981). Fossil specimens of P. cataracta have also been found in the unusual habitat of gypsum quarry sink hole ponds in East Milford, Nova
Scotia dating back to the Pre-Wisconsinan, approximately 84,000 years BP (Godfrey-
Smith et a!., 2003).
Phenotypic plasticity has been well documented among the Unionids, and many species, including P. cataracta and P. fragilis, display high intraspecific variation in conchological characteristics. Ortmann's law of stream position (1920) describes how shell shape and morphology can reflect habitat conditions: mussels from smaller streams tend to be compressed or flat whereas mussels from larger streams or rivers tend to be convex and swollen. Several Anodonta species as well as Pyganodon {-Anodonta) grandis have been shown to follow Ortmann's law of stream distribution where depth and height of shells increases as one goes further downstream (Mackie and Topping,
1988).
Few studies have explored the historical biogeography of unionids in Atlantic
Canada. Using allozyme data, Kat and Davis (1984) examined eight species of freshwater mussels in Nova Scotia in an attempt to elucidate potential colonization routes into the province. Based on this study, populations of Elliptio complanata were thought to have invaded Nova Scotia through the Isthmus of Chignecto and subsequently split into two groups colonizing northern and southern regions of the province. At the time of this study P. cataracta and P. fragilis were considered subspecies and were referred to as
12 P. c. cataracta and P. c. fragilis, respectively. P. c. cataracta and P. c. fragilis where shown to exhibit low levels of heterozygosity, polymorphism and variability in allele frequencies within Nova Scotia intimating that dispersal and colonization may have occurred by utilizing anadromous fish species as hosts (Kat and Davis, 1984). The use of anadromous fish species as hosts provides mussels the opportunity to be introduced into new habitats and will increase gene flow among geographically separated populations
(Kat and Davis, 1984). An earlier study by Kat (1983) comparing stomach morphology of P. c. cataracta from New Jersey and P. c. fragilis from Nova Scotia, led him to propose that the latter closely resembled the Eurasian Anodonta cygnea and therefore that
P. c. fragilis may represent a taxon with European ancestry which survived in a local refugium and is currently expanding its range into southern Nova Scotia (Kat, 1983).
As can be deduced from this brief summary of the studies conducted to date on these taxa, further phylogeographic studies are required to verify potential colonization routes and to establish historical and biogeographic patterns effecting current species distributions of Atlantic freshwater mussels. Patterns of genetic variability and diversity can provide insight into the historical biogeography of a species (Bermingham and
Moritz, 1998 and ref therein). Molecular techniques examining population genetic structure can provide insight into phylogeographic patterns and in turn can contribute valuable information for effective conservation programs of mussels (King et al., 1999).
13 Molecular markers
Given the difficulties in delineating Pyganodon cataract a and Pyganodon fragilis based on morphological traits and allozyme data, DNA-based molecular markers may be extremely useful for classifying their taxonomic status. Many conservation efforts have been hampered by incorrect and outdated taxonomic classifications based on morphology
(Avise, 1989; Daugherty et al, 1990; May, 1990; O' Brian and Mayr, 1991; Funk et al,
2002). The accurate identification of organisms and knowledge of their systematic relationships is a critical component to successful conservation and management strategies that aim to preserve biodiversity (e.g., Funk et al., 2002; Mace, 2004). With the use of molecular techniques such as DNA sequencing and Amplified Fragment
Length Polymorphisms (AFLPs), we will attempt to clarify some of these issues as they apply to the genus Pyganodon in Atlantic Canada.
Marine and freshwater bivalves exhibit a unique mode of mitochondrial DNA inheritance termed "double uniparental inheritance" or DUI (Skibinski et al., 1994;
Zouros et al., 1994; Hoeh et al., 1996a). Normally, animal mitochondrial DNA is inherited maternally, however, in species with DUI females transmit their mtDNA to both sons and daughters while males transmit their mtDNA to only their sons; therefore, females are homoplasmic and males are heteroplasmic for their mtDNA (Skibinski et al.,
1994; Zouros et al., 1994). Somatic tissue will normally contain predominantly the female (F-type) mtDNA whereas male (M-type) mtDNA is restricted to the spermatogenic tissues (Stewart et al., 1995). The apparent high fidelity of male and
14 female mitochondrial genomes provides two independent mtDNA markers to estimate phylogeny and gene flow among bivalves (Hoeh et al., 2002; Krebs, 2004; Riginos et al.,
2004). Furthermore, hybridization between species could be detected because males should contain mtDNA of both hybridizing species. However, due to difficulties of isolating and amplifying male mtDNA from these species, the male mitochondrial marker has not been included in our analysis.
Mitochondrial DNA has proven to be an extremely useful genetic marker for addressing systematic and phylogeographic patterns due its high rate of mutation (Avise,
2004). The cytochrome C oxidase subunit I gene, (abbreviated as COI or Coxl), within the mitochondrial genome, shows a relatively high level of diversity, and can be extremely useful in describing and differentiating cryptic species (Cox and Hebert, 2001;
Wares and Cunningham, 2001; Hebert et al., 2003). The COI gene is particularly valuable due to the availability of universal primers for most animal phyla (e.g., Folmer et ah, 1994) and has been recognized as the gene of choice for the Consortium for the
Barcode of Life Project (see http://www.barcoding.si.edu/) which aims to identify all species through the use of DNA barcodes, specifically the COI gene for animals (Hebert et ah, 2003). However, due to recent evidence suggesting the possibility of recombination in animal mitochondrial genomes (e.g., Lunt and Hyman, 1997; Tsaousis et al, 2005) and the possibility of mitochondrial introgression among species (e.g. Shafer and Stewart, 2007), it is becoming increasingly clear that DNA markers from both the nuclear and mitochondrial genomes should be used for phylogenetic analyses. One candidate nuclear gene for helping to resolve phylogenetic questions is the noncoding
15 internal transcribed spacer (ITS) region located between the 5.8S and 18S ribosomal
DNA genes (Palumbi, 1996). The ITS gene was found to be more variable than the mtDNA COI gene at the intraspecific level in the freshwater mussel the green floater,
Lasmigona subviridis (King et al., 1999), and could prove useful in investigating intraspecific variation within P. cataracta and P. fragilis.
Amplified fragment length polymorphisms or AFLP's are a category of PCR based multi-locus markers that screen different DNA regions located randomly throughout the nuclear genome (Vos et al., 1995; Bensch and Akesson, 2005). In contrast to microsatellite markers, which usually have to be developed and optimized for each species, or random amplified polymorphic DNA (RADPs), which may not be readily reproducible, AFLPs provide a reliable and rapid technique for assessing genetic diversity, population structure, and phylogeographic history (Mueller and Wolfenbarger,
1999). AFLP markers have broad taxonomic applicability and are found to be particularly useful for investigating population structure and differentiation at the species level, as well as having the ability to detect introgression and hybridization (Mueller and
Wolfenbarger, 1999). For these reasons, AFLP markers may be the method of choice for distinguishing between the putative taxa P. cataracta and P. fragilis. In combination, these molecular techniques will allow us to assess the taxonomic relationships of these freshwater mussels in Atlantic Canada.
The primary objectives of this research are to verify the taxonomic status and geographic distribution of Pyganodon cataracta and Pyganodon fragilis in New
Brunswick, Nova Scotia, Newfoundland and Prince Edward Island. Specifically, we will
16 try to answer the following questions: (1) are P. cataract a and P. fragilis two distinct species, (2) just one species or (3) a mixed, hybridizing population? Verifying the taxonomic status and distribution ofPyganodon cataracta and Pyganodon fragilis within
Atlantic Canada is an important first step in identifying species, evolutionary significant units or management units in need of protection (Ryder, 1986; Moritz, 1994). By combining morphological, mitochondrial, and nuclear sequence data in addition to a multi locus nuclear marker (i.e., AFLPs), we will address the taxonomic uncertainties surrounding P. cataracta and P. fragilis in Atlantic Canada. Lastly, the concluding chapter will comment on the value of utilizing molecular markers in conservation and systematic studies and provide general recommendations for the conservation of freshwater mussels in Atlantic Canada.
17 Chapter 2: Population structure and taxonomic status of Pyganodon cataracta and P. fragilis inferred from AFLP and sequence data
Introduction
The drastic decline of freshwater mussels (Bivalvia: Unionidae) in North America has been well documented (Bogan, 1993; Williams et al., 1993; Lydeard et ah, 2004).
Despite reaching their highest global diversity in North America, unionids are nonetheless regarded as one of the most endangered group of organisms on this continent
(Master et al., 1998; Strayer et al., 2004). Mussels in Canada are also highly imperiled, with over 50% of species identified as potentially at risk (Metcalfe-Smith and Cudmore-
Vokey, 2004). Anthropogenic factors in addition to their unique life history traits are the primary threats facing these animals (Bogan, 1993; Williams et al., 1993). Freshwater mussels are long lived and have limited mobility making them particularly vulnerable to changes in their environment (Kelly and Rhymer, 2005). As obligate parasites, their larvae (called glochidia) depend on the availability of a suitable fish host to complete their life cycle and to aid in their dispersal (Neves et al., 1985). As a consequence, factors that impact their fish hosts also impact these mussels.
Further complicating conservation efforts is the taxonomic uncertainty surrounding many unionid taxa due to classifications based solely on shell morphology and/or internal anatomy which are known to exhibit a high degree of phenotypic
18 plasticity (Kat, 1983; Williams and Mulvey, 1997; Lydeard and Roe, 1998; King et al,
1999). The use of molecular genetic markers has the potential to resolve the evolutionary relationships among closely related and/or cryptic taxa (Avise, 2004) and accordingly, a number of genetic studies have helped clarify the taxonomic and phylogenetic classification of unionids (Mulvey et al, 1998; Roe and Lydeard, 1998; King et al, 1999;
Roe et al, 2001; Baker et al, 2004).
Pyganodon cataracta, the eastern floater, and P. fragilis, the Newfoundland floater are two closely related and morphologically similar species whose taxonomic status has been a subject of continued debate (Clark and Rick, 1963; Kat, 1983,1986;
Hoeh, 1990; Cyr et al, 2007). These taxa have alternated between species and subspecies with the most recent evidence supporting the recognition of P. fragilis as a unique species based on preliminary allozyme and morphological data (Hoeh, 1990).
Allozyme data of five P. cataracta specimens from Pennsylvania and five P. fragilis specimens from Newfoundland were compared and were found to have one of the lowest observable genetic distances among 13 species of Pyganodon and Anodonta (Hoeh,
1990). Apart from adult P. cataracta being larger in size than P. fragilis, the only other distinguishing features used to differentiate them has been the colour of the periostracum and the presence of a double loop on the umbone of the former and a single loop on the latter (Clarke, 1981). A study by Hanlon and Smith (1999) suggested that many morphological characteristics used to distinguish P. cataracta and P. fragilis were unreliable thus highlighting the necessity of utilizing molecular markers in addition to morphology for delineating closely related and possibly cryptic species. Although a
19 recent study of Pyganodon spp. in Quebec by Cyr et al., (2007) found extremely low sequence divergence values for both mitochondrial and nuclear sequence data, these authors nevertheless argued in favour of retaining P. cataracta and P. fragilis as distinct species but, recognized the need for more comprehensive genetic studies incorporating the nuclear genome (Cyr et ah, 2007).
The exact distribution of these species in Atlantic Canada also remains unclear.
P. cataracta is fairly common and widespread throughout the southeastern United States and Atlantic Canada with confirmed populations in Quebec, New Brunswick, Nova
Scotia and Prince Edward Island (Clarke, 1981; Metcalfe-Smith and Cudmore-Vokey,
2004). It is the distribution of P. fragilis that remains particularly contentious in Atlantic
Canada. P. fragilis is thought to occur primarily in Newfoundland (Clarke, 1981; Hoeh,
1990), with suspected but unconfirmed distributions in New Brunswick and Nova Scotia and putative populations within Quebec (Metcalfe-Smith and Cudmore-Vokey, 2004).
Specimens exhibiting intermediate characteristics between P. cataracta and P. fragilis lead Clarke and Rick (1963) to suspect that hybridization may be occurring where their ranges potentially overlap. While no confirmed records of P. fragilis exist for New
Brunswick, some species resembling P. fragilis from Newfoundland have been encountered by Don McAlpine (pers. comm.) from various locations within the province.
Although historically P. cataracta were not believed to be present in Newfoundland or
Labrador, some specimens have been found which may possibly represent P. cataracta or one or more closely related Pyganodon species (John Maunder, pers. comm.). In addition, two separate infestation peaks of glochidia in January and April have been
20 observed in a single year on three-spined stickleback in Newfoundland (Threlfall, 1986) which may indicate the presence of two cryptic species that spawn at different times of the year (John Maunder, pers. comm.).
Despite the taxonomic uncertainties surrounding P. fragilis and P. cataracta, few studies have attempted to examine the population genetic structure and determine the distribution of these species in Atlantic Canada. P. cataracta and P. fragilis may represent distinct yet cryptic species that could be present in all four provinces of Atlantic
Canada but have, as of yet, not been definitively recognized due to subtle morphological characteristics and relatively limited mussel surveys. Newfoundland with its unique habitat, remote location and limited freshwater fish species has the potential to harbour distinct Pyganodon entities not yet described or discovered in the province.
Determining which species concept to adopt and which specific criteria to use when inferring species boundaries is critical in systematic studies. According to de
Queiroz (1998), all definitions of species currently in use are simply variants of a single concept that emphasizes species as distinct evolutionary lineages. Consequently, de
Queiroz (1998) argues that utilizing a combination of criteria incorporating different species concepts would be more useful than relying on any one single criterion. With this is mind we will primarily employ aspects of two common species concepts - the phylogenetic and genetic species concepts to delineate the taxonomic status of P. cataracta and P. fragilis. The essence of the phylogenetic species concept is that distinct species are monophyletic groups, consisting of all descendants of a common ancestor
21 (Rosen, 1979; Donoghue, 1985; Mishler, 1985). The genetic species concept recognizes species as groups or clusters of genetically distinct, non-interbreeding and isolated populations and the authors of this concept (i.e., Bradley and Baker, 2001 and Baker and
Bradley, 2006) emphasize the utility of molecular data such as DNA sequences for detecting morphologically cryptic genetic species.
In this study, we aim to determine the phylogeographic and population genetic structure of Pyganodon populations in an effort to resolve the taxonomic status and distribution of P. cataracta and P. fragilis in Atlantic Canada. To ascertain the taxonomic status of these putative species we combine morphological data together with amplified fragment length polymorphism (AFLP) data and sequence data for the first subunit of the mitochondrial cytochrome c oxidase (COI) region and the nuclear internal transcribed spacer region (ITS-1). Utilizing a nuclear multi locus marker, such as
AFLPs, in combination with mitochondrial and nuclear sequence data should provide a comprehensive picture of the population genetic structure of Pyganodon populations in
Atlantic Canada.
Methods
Sample collection and DNA isolation
All Pyganodon specimens from Nova Scotia and New Brunswick were collected by hand through direct observation in shallow waters and were stored on ice in the field and subsequently preserved in 95% ethanol. For locations exhibiting low populations
22 densities, only 1-5 voucher specimens were collected for DNA analysis. Sampling location names from Newfoundland, Nova Scotia, and New Brunswick are provided in
Figure 2.1 and Table 2.1. All specimens will be donated to and catalogued at the New
Brunswick Museum or the Acadia University Wildlife Museum to aid in future genetic and morphological studies of Pyganodon species in Atlantic Canada. Mussels were collected if they exhibited general characteristics of the genus Pyganodon. Identifying morphological features of the genus Pyganodon include inflated umbones on older and larger individuals, lack of hinge teeth and relatively thin fragile shells (Hoeh, 1990). In addition to specimens of the genus Pyganodon, Anodonta implicata, Elliptio complanata, and Margaritifera margaritifera were also periodically collected and these mussels were returned to the water.
A series of Pyganodon samples from Newfoundland was collected by Randy
Hoeh and John Maunder in July 1994. Samples were collected by either visual surveys in shallow water or by snorkeling and were kept on ice in the field and were stored in a
-80°C freezer until subsequent dissection and DNA analysis in March 2007. Samples used for DNA analysis were chosen based on the geographic distribution (e.g., the southwest, northwest, northeast and southeast "corners") of Newfoundland and incorporating both northern and southern subdrainages of the province. Within the northern subdrainage mussels were sampled from Joe Farrels Pond and Bonne Bay from the Northern Peninsula, First Pond and Notre Dame from the Notre Dame district as well as Noel's Pond from St. George's district for a total of 29 individuals. From the southern subdrainage, 24 mussels were sampled from Big Pond and Red Rocks from the southern
23 coast, L'Anse-au-Loop from the Burin Peninsula, Bird's Pond from the Avalon
Peninsula, as well as Cordoy Pond from St. George's district (Table 2.1).
Based on historical records obtained from the Nova Scotia Museum curatorial report (Davis, 2004) known populations of Pyganodon were surveyed from May to July
2007 in two out of the three major drainage areas from the province of Nova Scotia: The
Bay of Fundy, Gulf of St. Lawrence drainage and Cape Breton drainage area. Not included in our analysis was the southeastern Atlantic drainage area. Within the Bay of
Fundy and Gulf of St. Lawrence drainage, mussels were sampled from Shey Lake,
Savage Lake, Pigott Lake and Murphy Lake in Hants County as well as Miner's Meadow
Pond and Belcher Street Pond in Kings County for a total of 12 individuals. In addition, eleven individuals also within the Bay if Fundy and Gulf of St. Lawrence drainage area were collected at the following sites: Vickery Lake, Angevine Lake, Tidd Pool and
Truemanville Pond from Cumberland County. Finally, from the Cape Breton drainage basin, a total of 22 individuals were sampled from Lake Ainslie, Hays River and Lake
O'Law in Inverness county and Blacketts Lake and Gillis Lake from Cape Breton County
(Table 2.1). Four Pyganodon populations were newly discovered in Nova Scotia including Belcher Street Pond and Murphy Lake in Hants County as well as
Truemanville Pond and Tidd Pool in Cumberland County.
New Brunswick localities were selected in consultation with Don McAlpine from the New Brunswick Natural History Museum as well as the Nova Scotia Curatorial report
(Davis, 2004). Both major drainage areas were sampled for Pyganodon in the Province
24 of New Brunswick. Within the Saint John and southern Bay of Fundy drainage system mussels were collected from Baker Lake and Lac a Lang from Madawaska County, Lake
Utopia from Charlotte County and Darlings Lake from Kings County for a total of 15 individuals. Eight individuals within the Gulf of St. Lawrence and northern Bay of
Fundy drainage area were collected from Poucette Lake and Greer Lake representing
Westmorland and Northumberland Counties, respectively. One additional population,
Wild Goose Lake in Madawaska County, from extreme northwestern New Brunswick is unique in that it is the only area of New Brunswick that is part of the northern Gaspe
Peninsula drainage area within the larger St. Lawrence River estuary drainage system.
Four individuals were collected from this location. In addition to the samples listed above, three specimens from Winter River, Prince Edward Island were collected in
August 2007 and donated by Don McAlpine from the New Brunswick Museum.
25 Figure 2.1. Geographic distribution oiPyganodon sampling locations in Atlantic Canada.
26 Table 2.1. Description of sampling localities by province, subdrainage area and site location of Pyganodon collected, including sample size for the COI, ITS-1 and AFLP analysis. Sample Size Province Subdrainage area Site location COI ITS-1 AFLPs
Newfoundland Northern Newfoundland (ND) First Pond, Notre Dame Bay 8 2 Notre Dame, Notre Dame Bay 7 1 2 Joe Farrels Pond, Northern Peninsula 8 2 2 Bonne Bay, Northern Peninsula 5 — - Noel's Pond, St. George 4 2 2 Southern Newfoundland (SD) Cordoy Pond, St. Georges 9 2 2 Big Pond, South Coast 2 - Red Rocks, South Coast 2 - Bird's Pond, Avalon Peninsula 4 - - L'Anse-au-Loup, Burin Peninsula 4 — - Nova Scotia Bay of Fundy / Gulf of St. Lawrence (BF) Shey Lake, Hants 3 1 Murphy Lake, Hants 2 - Savage Lake, Hants 1 - Pigott Lake, Hants 2 1 Miner's Meadow, Kings 3 - Belcher St. Pond, Kings 1 - Truemanville pond, Cumberland 4 - Tidd Pool, Cumberland 4 1 Vickery Lake, Cumberland 2 - Angevine Lake, Cumberland 1 - Cape Breton (CB) Lake Ainslie, Inverness 2 - Hays River, Inverness 5 1 Lake O'Law, Inverness 5 - Blacketts Lake, Cape Breton 5 - Gillis Lake, Cape Breton 5 1 Table 2.1. Continued Sample Size Province Subdrainage area Site Location COI ITS-1 AFLPs
New St. John/ S. Bay of Fundy (SJ) Baker Lake, Madawaska Brunswick Lac a Lang, Madawaska 5 1 1 Lake Utopia, Charolette 5 2 3 Darlings Lake, Kings 1 — - St. Lawrence / N. Bay of Fundy (SL) Poucette Lake, Westmorland 5 1 1 Greer Lake, Northumberland 3 - - St. Lawrence River Estuary- Northern Wild Goose Lake, Madawaska 4 - 2 Gaspe Peninsula (GP) Prince Edward Prince Edward Is. (PE) Winter River, Queens Is. All specimens were labeled, measured and stored in 95% ethanol for subsequent
DNA analysis. Genomic DNA was isolated from a small portion of somatic (mantle) tissue using a Qiagen DNeasy tissue kit following the manufacturer's protocol. All mantle tissue previously stored in 95% ethanol was dried in a 37 C incubator overnight prior to extraction. Gonad tissues were specifically avoided to prevent possible
"contamination" with male mtDNA due to the presence of the system of double uniparental inheritance of mitochondrial DNA in a number of bivalves including unionids
(e.g. Hoeh et al., 1996b). DNA quality was assessed visually following electrophoresis of a small aliquot of total DNA in a 1% gel stained with ethidium bromide.
Morphological analysis
Morphological characteristics commonly used to differentiate P. cataracta from
P.fragilis were examined to determine if these conchological traits are in fact useful in distinguishing these two putative species. All mussels identified with genetic markers from Nova Scotia and New Brunswick were examined morphologically, with the exception of four New Brunswick specimens donated by Don McAlpine from the New
Brunswick Natural History Museum. Only tissue samples were donated and readily available for DNA analysis therefore, the shell measurements of the aforementioned museum specimens as well as the Newfoundland mussels could not be included in this part of the study.
29 In total 65 individuals were included in the morphological analysis.
Conchological measurements of shell length (SL), shell height at umbone (SH), and shell width (SW) were taken using standard vernier calipers (accuracy of 0.1 mm). Ordinal characters such as colour of periostracum (CP), colour of nacre (CN), presence or absence of discolouration on nacre (DC), number of growth rests (NG), appearance of green rays (GR), presence of a double loop structure (DL), and extent of erosion (EE) where also included in the analysis. Another characteristic used to differentiate P. cataracta and P. fragilis is the shape of the posterior end of the shell being either rounded or blunt, respectively (see Figure 1.2a). However, this description was extremely subjective and was therefore excluded from the analysis because it could not be accurately identified as being one shape or the other in most specimens examined.
Quantitative measurements and ordinal data were transformed using an extension of Gower's (1971) general coefficient of similarity as modified by Podani (1999), which allows for cluster analyses or multidimensional scaling of mixed data types. A principle components analysis as well as a UPGMA dendogram was performed in the SYN-TAX
2000 program (Podani, 2001).
DNA sequencing and data analysis
The F-type cytochrome oxidase subunit I (COI) mitochondrial gene was amplified using the universal COI primers designed by Folmer et a/.(1994), modified by Walker et al. (2006), LCO-22me2 (5'-GGTCAACAAAYCATAARGATAT-3') and HCO-700dy2
(5'-TCAGGGTGACCAAAAAAYCA-3') via the polymerase chain reaction (PCR).
30 Amplification reactions contained ~ lOOng of genomic DNA, 1 X PCR buffer (Promega
GoTaq), 2.5mM MgCl2, 0.4mM dNTP's, l.OuM of each primer and 0.625U GoTaq DNA polymerase (Promega Corporation; Madison, WI, USA) in a total volume of 25ul. PCR reactions were carried out in a DNA engine PTC-200 thermal cycler (MJ Research) under
o o the following conditions: initial denaturing at 94 C for 2 minutes; 40 cycles of 94 C
o o denaturing for 45 seconds, 48 C annealing for 45 seconds, 72 C extension for 45 seconds, with a final 4 minute extension at 72 C. The COI amplicons were viewed on a 1% agarose gel in 1 x TAE buffer and were purified using a gel extraction kit (Qiagen).
The internal transcribed spacer region (ITS-1) gene separating 5.8S and 18S ribosomal DNA genes was amplified via the PCR using the primers ITS-1 18S (5'-
AGCTTGCTGCGTTCTTCATCG-3') and ITS-1 5.8S (5'-
AAAAAGCTTCCGTAGGTGAAC-3') (King et al, 1999). PCR reactions and purifications were similar to the protocols described for the COI gene with the following changes in cycling parameters: an initial 2 minute denaturing at 94 C, 40 cycles of
94 C for 30 seconds, 52 C for 30 seconds, 72 C for 1 minute and a final 5 minute
o extension at 72 C. Double strand sequencing of all purified PCR products was performed on an Applied Biosystems 3730x1 DNA analyzer system at McGill University and
Genome Quebec Innovation Centre Sequencing Platform. Approximately 575bp of sequence was obtained for both the COI and ITS-1 genes.
31 All sequences were aligned using BioEdit V.7.0.5.3 (Hall, 1999) and were visually confirmed prior to phylogenetic analysis in PAUP* 4.0b 10 (Swofford, 2001).
Maximum parsimony (MP) and neighbor-joining (NJ) methods were conducted to estimate the phylogenetic relationships among Pyganodon specimens from Atlantic
Canada for both the COI and ITS-1 regions separately using both rooted and unrooted trees. Sequences for outgroup species were obtained from GenBank and included
Pyganodon grandis (Accession nos. AF231734, AF156504) and Strophitus undulatus
(Accession nos. AF 156505) for the COI gene, as well as Pyganodon lacustris (sequence obtained from W.R. Hoeh) and Pyganodon grandis (Accession nos. EF488195,
EF488195) for the ITS-1 gene. All available ITS and COI sequences from putative specimens of P. cataracta and P.fragilis were also obtained from GenBank (Table 2.2).
Specimens that clustered with either of these sequences in the unrooted trees of all
Atlantic Canada Pyganodon samples were tentatively identified as "P. cataracta" or "P. fragilis", respectively. This working taxonomic hypothesis was used as a basis for investigating patterns of morphological differentiation and geographic structuring of
Pyganodon populations in Eastern Canada.
32 Table 2.2 List of reference specimens and outgroup species used in the COI and ITS-1 analysis, their locality and GenBank accession numbers.
GenBank accession number
Species Locality COI ITS-1
Pyganodon cataracta Quebec EF418016 EF488191 Quebec EF418023 EF488190 Pyganodon fragilis Quebec EF418017 EF488192 Newfoundland AF406800 Pyganodon grandis Michigan AF156504 Michigan AF231734 Quebec - EF488196 Quebec - EF488195 Strophitus undulatus Michigan AF156505
A minimum-spanning network of COI mtDNA haplotypes was created using the
TCS 1.21 program that implements statistical parsimony (Clement et al., 2000). The resulting COI haplotypes were also subjected to a maximum parsimony analysis using
PAUP (Swofford, 2001). MP analyses employed a full heuristic search with characters unweighted and unordered and tree bisection-reconnection branch swapping with 100 stepwise random addition replicates. A genetic distance matrix for the NJ analysis was created using Kimura's two-parameter model. (A simple distance model like Kimura's two-parameter model is appropriate in this instance given the extremely small genetic divergence levels- see below). Inter-specific and intra-specific genetic differences between identified clades and outgroup species were also calculated using Kimura's two- parameter model in MEGA 3 (Kumar et ah, 2004). In addition, a maximum likelihood
33 (ML) analysis was performed. The best evolutionary model for a ML analysis was determined using the program MODELTEST 3.6 (Posada and Crandall, 1998) and searches were consequently conducted using the HKY 85 model (Hasegawa et al., 1985).
All the resulting trees were evaluated using 1000 bootstrap replicates for the MP and NJ analyses and 100 replications for the ML analysis on 575bp of both COI haplotype and
ITS-1 sequence data separately.
An analysis of molecular variance (AMOVA, Excoffier et al., 1992) on the COI sequence data was performed using Arlequin version 3.1 (Excoffier et al., 2005) to examine the partitioning of genetic variance among provinces and the putative species
(i.e. the "P. cataracta" and "P. fragilis" clades) represented in the unrooted NJ and MP analyses. AMOVAs were conducted on pairwise squared Euclidean distances with a significance level assessed by building a null distribution of distance values created by
1000 permutations (see Excoffier et al., 1992).
AFLPprocedure and data analysis
Amplified fragment length polymorphism bands (AFLPs) were analyzed for subset of 35 individuals collected from Newfoundland (n=12), Nova Scotia (n=15), and
New Brunswick (n=8) using a modified version of the Vos et al., (1995) procedure. For the AFLP analysis 13 "P. cataracta" and 22 "P. fragilis" samples were arbitrarily chosen to represent all three provinces. Approximately 500ng of genomic DNA was digested with 5U of both EcoRl (New England Biolabs [NEB]) and Msel (NEB), lx NEB buffer
34 supplemented with 5ug of BSA in a total volume of 50ul. Samples were incubated at
37°C for 3 hours followed by enzyme inactivation at 70°C for 15 minutes. Ligation reactions consisted of 50ul of the digested DNA reaction together with a lOul solution containing 5uM iscoRI-adaptors, 50uM Msel-adapters (see Table 2.3), 1U T4 DNA- ligase (Invitrogen) and 5x T4 DNA ligase buffer (Invitrogen) incubated at 5°C overnight.
Preselective PCR reactions contained 3ul of the ligated DNA reaction, 1.5mM MgCb,
0.24mM each dNTP, 0.2nM of primers EcoRl + A and Msel + C, 0.5U of GoTaq
(Promega) and 10X PCR buffer in a total volume of 25ul. PCR cycling parameters were as follows: initial denaturing at 94°C for 2 minutes, 30 cycles of 94°C for 30 seconds,
56°C for 30 seconds, 72°C for 2 minutes and a final 10 minute extension at 60°C. Pre- amplification reactions were diluted with lOOul of 1 x TE (ImM tris-HCl, ImM EDTA, pH 7.6) and were used for all successive selective PCR reactions or amplifications using a total of eight primer combinations (Table 2.3). Selective PCR reactions were carried out in a final volume of 12.5ul and included 2.5(0.1 of the diluted pre-amplification reactions, 2mM MgCl2, 0.24mM of each dNTP, 0.2uM of Msel + 3 primer, 0.04uM labeled EcoRI + 3 primer, lug BSA, 0.5U GoTaq (Promega) and lOx PCR buffer. The selective amplification consisted of an initial 2 minute denaturing period at 94°C, 12 cycles of 94°C for 30 seconds, 65 °C for 30 seconds (reducing the annealing temperature by 0.7°C per cycle) and 72°C for 2 minutes. This cycling procedure was followed by an additional 23 cycles of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 2 minutes with an additional extension period of 72° for 10 minutes.
35 Table 2.3. Adapters and selected primer sequences and combinations used in the AFLP analysis.
Adapters EcoRl 5'- CTCGTAGACTGCGTACC-3' 3 '-CATCTGACGC ATGGTTAA-5' Msel 5'-GACGATGAGTCCTGAG-3' 3 '-TACTCAGGACTCAT-5'
Primer Sequence (5'-3') EcoRl+A GACTGCGTACCAATTCA Msel+C GATGAGTCCTGAGTAAC EcoRl+ACC GACTGCGTACCAATTCACC EcoRl+AGG GACTGCGTACCAATTCAGG Msel+C A A GATGAGTCCTGAGTAACAA Msel+C AG GATGAGTCCTGAGTAACAG Msel+C AT GATGAGTCCTGAGTAACAT Msel+CTC GATGAGTCCTGAGTAACTC Msel+CTG GATGAGTCCTGAGTAACTG
Combinations 1 Eco+ACC-Mse+CAA 2 Eco+AGG-•Mse+CAT 3 Eco+AGG-•Mse+CAA 4 Eco+AGG-•Mse+CTG 5 Eco+ACC-Mse+CTC 6 Eco+AGG-•Mse+CAG 7 Eco+ACC-Mse+CAG 8 Eco+AGG-•Mse+CTC
AFLP products were denatured at 90°C for 3 minutes and then cooled on ice. A
3ul aliquot of each sample was loaded onto either a 6 % Long Ranger (Amresco Inc.) or
7% long-read polyacrylamide gel (Amersham Biosciences) and was run on an
ALFexpress II automated DNA fragment analyzer (Amersham Biosciences). Each
36 sample included an internal sizer of 50-500bp and in addition three lanes of every run contained only external sizers to aid in accurate calibration of bands sizes.
ALFwin Fragment Analyzer software (Amersham Biosciences) was used to visualize and score gel images. For each primer combination, individuals were scored based on presence (1) or absence (0) of bands ranging from 60-270bp. Five individuals were run on separate gels for each primer combination to assess reproducibility of banding patterns and all individuals were scored without reference to sample number or species identity. Binary presence/absence data were converted into a pairwise distance matrix using the Jaccard coefficient (Jaccard, 1908) in the statistical program R version
2.4.1. The pairwise distance matrix was then used to generate an unroooted neighbor- joining (NJ) tree in MEGA 3.1 (Kumar, 2004). In addition, the TREECON program
(Van de Peer and de Wachter, 1994) was also used for the NJ analysis because it is capable of producing bootstrap values for binary data. This software program uses a modified version of the Jaccard coefficient called the Link et al., (1995) method to create a distance matrix for the NJ analysis.
The genetic structure of Atlantic Pyganodon populations was also investigated using the STRUCTURE program version 2.1 (Prichard et al., 2000) and Arlequin version
3.1 (Excoffier et al., 2005). The ALFPdat program (Ehrich, 2006) was used to convert binary AFLP data into a data matrix for use in both STRUCTURE and Arlequin.
STRUCTURE uses Bayesian models to cluster individuals into genetically distinct populations. To estimate the number (K) of populations or clusters, five independent
37 runs of K from 1 to 10 were performed for 100,000 iterations with a 20,000 iteration burn-in period. The mean posterior probability of each K or the "log probability of the data," L (K), was calculated to determine the optimal K value. However, often the most likely value of K is not clearly defined, we therefore implemented the procedures described by Evanno et al., (2005) to compute the rate of change between consecutive K values (AK) as a means to identify the best estimate of K. The best estimate of K was selected by plotting the rate of change between consecutive K values (AK) on a graph and selecting the uppermost or highest value of this distribution as the best estimate of the number of clusters (K). All simulations in STRUCTURE were completed incorporating the admixture ancestry model and correlated allele frequency model with no prior information on population origin. Individuals were subsequently assigned to populations based on probability of membership (q) with values of q > 0.80 being sufficient to confidently assign an individual to a population and values of 0.20 < qgr0up < 0.80 representing an admixed population where an individual displays mixed ancestry belonging to more than one cluster.
An analysis of molecular variance (AMOVA, Excoffier et al., 1992) with the
AFLP data was also performed using Arlequin version 3.1 (Excoffier et al, 2005) following similar parameters as described for the sequence data above. For the AMOVA, individuals were partitioned into groups according to the three main geographic clusters identified by STRUCTURE, or alternatively into groups based on putative species inferred from the COI sequence data.
38 Results
Morphological analysis
The principle components analysis of common morphological characters failed to separate individuals from Nova Scotia and New Brunswick into two separate groups identified as "P.fragilis" and "P. cataracta" based on COI sequences (Figure 2.2). The first two principle components explained 70% of the variation observed. When examining both the biplot and the UPGMA dendogram (Figure 2.3), no trends or relationships could be distinguished among putative species. However, minor groupings of individuals were clustering together based on sampling location (i.e. province and/or subdrainage). A few Nova Scotia "P. cataracta''' individuals grouped together and a few
"P. fragilis" specimens from New Brunswick were also found to form a small cluster in the UPGMA dendogram. In essence, individuals tentatively identified as one or the other species are found throughout this PCA biplot and UPGMA dendogram.
39 • P. cataracta - NB P. fragilis - NB • P. cataracta - NS » P. fragilis - NS CM
• -1 • *
-3 I ' ' ' I I " * " I • -2 -1 0 1 a 3 Axis 1
Figure 2.2. Principle component analysis biplot showing the relationship between putative "P. cataracta" and "P. fragilis" individuals (according to their COI haplotype) based on common morphological measurements. Each square point on the graph represents a single individual and circles denote the conchological descriptions used in the analysis: shell length (SL), shell width (SW), shell height at umbone (SH), colour of periostracum (CP), colour of nacre (CN), presence or absence of discolouration on nacre (DC), number of growth rests(NG), appearance of green rays(GR), presence of a double loop structure(DL), and extent of erosion(EE). The length and direction of the lines associated with each conchological character indicates how strongly correlated these variables are with the first and second axes.
40 • P. cataracta - NB a P. fragilis - NB • P. cataracta - NS 0.36 i » P. fragilis - NS 0.34 0.32 0.3 0.28 0.2(5 0.24 0.22 1 02 | 0.18 .3 0.16 Q 0.141 0.12 0.1 0.08 0.06-j 0.04 0.02 0 •!1BHI!#«»#«M»™»
Figure 2.3. UPGMA dendogram based on common morphological characteristics of the presumptive "P. cataracta" and "P. fragilis" specimens from Nova Scotia and New Brunswick.
Few specimens examined morphologically reached over 90mm in length and although the maximum limit of P. fragilis is not expected to exceed this length, two out of three specimens over 90 mm were genetically identified as "P. fragilis" based on COI sequences. Furthermore, many individuals identified as "P. fragilis " were found to have a double loop which is a common characteristic used to identify P. cataracta.
Conversely, several presumed "P. cataracta" specimens were found to have a single loop. When examining the properties of the periostracum, the colour seemed to be associated more closely with substrate type rather than species descriptions. In general, specimens collected from soft sand or muddy substrates were shiny, bright green to straw
41 yellow in appearance while mussels collected from rocky or coarse sand substrate were dull brown to black in colour.
Mitochondrial and nuclear DNA sequencing analysis
Nucleotide sequences 575bp in length from the mitochondrial COI gene were obtained forl28 Pygandon specimens, representing 33 localities across Atlantic Canada.
From the sequences examined 14 sites were variable, of which only eight were phylogenetically informative. The transition to transversion substitution ratio was 11.8:1 with an overall nucleotide composition consisting of 41.1% thymine, 23.4% guanine,
19.2% adenine and 16.3%) cytosine.
Comparing our samples to reference specimens from GenBank, 42 and 86 individuals were putatively identified as "P. cataracta " and "P. fragilis ", respectively.
The minimum spanning network revealed nine "P. cataracta " and six "P. fragilis " haplotypes, with the former having two relatively common haplotypes (H7 and H8) and the latter with only one predominant haplotype (HI) (Figure 2.4). Approximately 62% of all 128 individuals were identified as Haplotype 1 and all other haplotypes differed from this main haplotype by one to four base pair changes. Only two transitional base pair changes separated P. cataracta and P. fragilis. No clear-cut separation of haplotypes by geographic region was observed as the three most common haplotypes from both presumptive species were represented in each province.
42 P. fragilis = Clade A P. cataracta = Clade B
H1 H7 79 21
Figure 2.4. Statistical parsimony network of P. fragilis and P. cataracta mtDNA haplotypes for the COI gene, where each circle represents a single haplotype and the frequency of the haplotype is indicated by the size of the circle (sample numbers are provided for n >3 individuals). Squares denote a 95% root probability; lines connecting haplotypes represent a single base pair difference and nodes signify an intermediate haplotype or two base changes.
The average interclade distances was 0.5% between clades A and B. In contrast, distances ranged from 4.7% to 15.9% when P. cataracta and P. fragilis were compared to the three outgroup species: P.lucustris, P. grandis and Strophitus undulatus (Table 2.4).
Due to low sequence divergence values, P. fragilis and P. cataracta will hereafter be commonly referred to as Clade A and Clade B, respectively.
43 Table 2.4. Genetic distances (Kimura two parameter model) of cytochrome C oxidase I sequences between outgroup species Strophitus undulatus, P. grandis, P. lacustris and P. cataracta and P. fragilis.
S. undulatus P. grandis P. lacustris P. cataracta P. fragilis S. undulatus — P. grandis 0.145 — P. lacustris 0.159 0.095 P. cataracta 0.126 0.096 0.048 P. fragilis 0.129 0.096 0.047 0.005
The neighbor-joining, maximum parsimony and maximum likelihood analyses of the COI sequence data generated nearly identical trees. The only observable difference in the NJ, MP and ML trees was that the rooted NJ tree grouped haplotypes 8 and 10 together with 59% bootstrap support (Figure 2.5). Unrooted and rooted NJ and MP trees also produced similar topologies although unrooted trees divided "P. fragilis " and "P. cataracta " into two distinct groups with relatively high (>79) bootstrap support (Figure
2.6). In contrast, when the sequences were rooted, only the "P. ft-agiHs"clad e was monophyletic; the remaining "P. cataracta" sequences formed an unresolved polytomy.
For purposes of discussion, P. fragilis and P. cataracta will be referred to as clades A and
B, respectively while recognizing that the P. cataracta specimens do not constitute a true monophyletic group based on the COI sequences. Although lower bootstrap support values were observed for the rooted MP tree (63%) (Figure 2.7), the unrooted MP analysis displayed relatively strong support for the division of clades A and B (79%). In general stronger, but only moderate, bootstrap support for the division of Clade A and B was observed in the NJ tree (74%) and ML analysis (69%) (Figure 2.8).
44 AMOVA analyses based on F- statistics were found to be significant (
P < 0.001) indicating genetic differentiation between clades A and B. Most of the variance (80.95%) was found among populations (i.e., between these two clades) as opposed to within populations (19.05%). When the AMOVA was repeated on the COI data with populations defined by province rather than by mtDNA clade, only moderately low levels of differentiation was observed among provinces (®ST=0.173, P <0.001) and most of the variation was distributed within (82.6%) rather than among provinces
(17.3%).
Hap1 (NL=43, NL=1*. NS=14, NB=19, PE=1,QC=1*) Hap2 (NS=3) ' ;
74 Hap3 (NS=2) Hap4 (NB=2) Hap5(NB=1) Hap6(NL=1) Hap7 (NS=14, NB=5, PE=2) Hap9 (NS=3) 99 Hap10(NS=2) 59 Hap8 (NL=14, NS=3, QC=1*) Hap11 (NS=1) Hap12(NS=1) 92 Hap13(NS=1) Hap14(NS=1) Hap15 (QC=1*) P. lacustris P. grandis 100 P. grandis Strophitus undulatus Figure 2.5. 50% majority rule consensus tree (rooted) produced by a neighbor-joining analysis of the cytochrome c oxidase subunit 1 (COI) sequence data (totaling 575bp) from 128 Pyganodon specimens. Bootstrap values are based on 1000 replicates. Haplotype numbers are followed by number of individuals from each locality. (* denotes reference and outgroup samples obtained from GenBank and f denotes sample obtained from W.R. Hoeh).
45 • Hap7 (NS=14, NB=5, PE=2) •Hap8(NL=9, NS=3, QC=1*) Hap9 (NS=3) Hap10 (NS=3) 81/79 Clade B Hap11 (NS=1) P.cataracta •Hap12(NS=1) •Hap13(NS=1)
• Hap14 (NS=1) • Hap15 (QC=1*)
• Hap1 (NL=43, NL=1*, NS=14, NB-19 PE=1,QC=T) Hap2 (NS=3) Hap3 (NS=2) Clade A • Hap4 (NB=2) P. fragilis • Hap5 (NB=1) . Hap6(NL=1)
Figure 2.6. 50% majority rule consensus tree (unrooted) produced by neighbor-joining and maximum parsimony analyses of the cytochrome c oxidase subunit 1 (COI) sequence data (totaling 575bp) from 128 Pyganodon specimens. Bootstrap values of both phylogenetic methods (NJ/MP) were based on 1000 replicates. Haplotype numbers are followed by number of individuals from each locality.
46 Hap1 (NL=43, NL=1*, NS=14, NB=19, PE=1,QC=1*) Hap2 (NS=3)
Hap3 (NS=2) 63 Hap4 (NB=2)
Hap5(NB=1)
Hap6NL=1)
Hap7 (NS=14, NB=5, PE=2)
Hap8(NL=9, NS=3,QC=1*)
75 Hap9 (NS=3)
Hap10(NS=3)
Hap11 (NS=1)
Hap12(NS=1) 88 Hap13(NS=1)
Hap14(NS=1)
Hap15(QC=T)
P. lacustris
P. grandis 100 P. grandis
Strophitus undulatus
Figure 2.7. 50% majority rule consensus tree (rooted) produced by a maximum parsimony analysis of the (COI) sequence data (totaling 575bp) from 128 Pyganodon specimens. Bootstrap values are based on 1000 replicates. Haplotype numbers are followed by number of individuals from each locality. (* denotes reference and outgroup samples obtained fro GenBank and f denotes sample obtained from W.R. Hoeh)
47 Hap1 (NL=43, NL=1*, NS=14, NB=19, PE=1,QC=1*)
Hap2 (NS=3)
Hap3 (NS=2) 69
Hap4 (NB=2)
Hap5(NB=1)
Hap6(NL=1)
Hap7(NS=14), NB=5, PE=2)
Hap8(NL=9, NS=3,QC=1*)
83 Hap9 (NS=3)
Hap10(NS=3)
Hap11 (NS=1)
Hap12(NS=1)
Hap13(NS=1)
Hap14(NS=1)
Hap15(QC=1*)
P. grandis 100 P. grandis
P. lacustris
Strophitus undulatus
Figure 2.8. 50% majority rule consensus tree (rooted) produced by a maximum likelihood analysis of the (COI) sequence data (totaling 575bp) from 128 Pyganodon specimens. Bootstrap values are based on 100 replicates. Haplotype numbers are followed by number of individuals from each locality. (* denotes reference and outgroup samples obtained fro GenBank and f denotes sample obtained from W.R. Hoeh)
48 Nucleotide sequences 575bp in length from the nuclear ITS-1 gene were obtained for 20 Pyganodon specimens, representing 16 populations from all four provinces in
Atlantic Canada. All 20 sequences, comprising a mixture of'?, cataracta" (n=9) and
"P. fragilis " (n=l 1) specimens, were identical with no variable sites. A NJ and MP analysis produced congruent phylogenetic trees identifying only one clade among specimens examined with high bootstrap support (Figure 2.9). One variable site was found within one P. cataracta sample obtained from GenBank (Accession no.
EF488191), however, because these specimens contained no phylogenetically informative information, no further analyses were conducted.
AFLP analysis
AFLP analysis of Pyganodon populations from Newfoundland, Nova Scotia and
New Brunswick yielded a total of 235 loci, 222 of which were polymorphic. Nova Scotia exhibited the highest proportion of polymorphic sites (n=91) followed by New
Brunswick (n=66) and Newfoundland (n=65). When looking at differences among the two clades, clade A or "P. fragilis" contained 110 polymorphic loci and clade B or "P.. cataracta " had 80 polymorphic sites for a total of 190.
A neighbor-joining analysis failed to separate clades A and B; instead three main groups were identified that were loosely separated by geographic distribution (Figure
2.10) but with relatively low bootstrap support. Barring a few exceptions most specimens grouped together by province and no discernable patterns were apparent among subdrainages.
49 H2281 (NL) L01 (NS) H2326 (NL) H2274 (NL) L19(NS) L149(NB) L354a (PE) L220 (NB) H2267 (NL) L133(NS) H2280 (NL) 91/89 L75 (NS) P. fragilis (QC*) L93 (NS) H2272 (NL) L197(NB) H2266 (NL) P. cataracta (QC*) L218(NB) L162 (NB) L354b (PE) L153(NB) P.cataracta (QC*) P. grandis (QC*) P. grandis (QC*)
Figure 2.9. 50% majority rule consensus tree produced by both a neighbor-joining and maximum parsimony analysis of the internal transcribed spacer region (ITS-1) sequence data (totaling 575bp) from 20 Pyganodon specimens. Bootstrap values of both phylogenetic methods (NJ/MP) were based on 1000 replicates. (* denotes reference and outgroup samples obtained from GenBank).
50 93 H2274 NLND (A) — H2280 NLSD (B) 67 H2281 NLSD(B) — H2350NLND(A) - H2272 NLND (A) H2164NLND(A) NL _ea -91. H2222 NLND (A) (+ some NS) - H2365 NLSD (A) — L06 NSBF (A) • L08 NSBF (A) • H2326 NLND (A) L19NSBF(A) L31 NSBF (A) •H2162NLND(A) L197NBSL(A) — L222 NBSJ (B) 67 •L153NBSJ(B) L162NBSJ(B) L218NBSJ(A) -L180NBGP(A) NB L178NBGP(A) ^ L220 NBSJ (B) (+1NL) 76 — L90 NSCB (B) L93 NSCB (B) • L41 NSBF (B) H2266NLSD(A) H2267 NLSD (A) L01 NSBF (B) L87 NSBF (A) • L55 NSBF (B) NS • L65-NSBF (A) L75 NSBF (A) (+ some NL) L133NSCB(A) L106NSCB(B) L122NSCB(B)
0.01
Figure 2.10. Neighbor-joining analysis of 35 Pyganodon specimens based on data from 235 AFLP loci with 100 bootstrap replicates. Specimen names are followed by locality (province and subdrainage) and clade (either A or B). Provincial abbreviations are NL (Newfoundland), NS (Nova Scotia), and NB (New Brunswick). Refer to table 2.1 for subdrainage abbreviations.
Clustering analysis in STRUCTURE identified three distinct populations.
Initially, the optimal K value was not clearly defined by using the log probability of the data (Figure 2.11 A) and therefore AK values were calculated, confirming that our data set contained three main clusters (Figure 2.1 IB).
51 A. L(K)
•2200
B. AK
Figure 2.11. Graphs depicting the methods used for determining value of K or number of groups from the AFLP data set (A) Mean L(K) values for 5 runs at each K value (1 to 10) (B) A K for our dataset where the true K is the highest point on the corresponding graph. Calculations followed those of Evanno et ah, (2005).
52 STRUCTURE assigned 26 individuals into clusters based on the three provinces included in the AFLP analysis (NL, NB or NS) with a high average proportion of membership (i.e., qgroup > 0.8) (Figure 2.12). The remaining nine specimens were assigned to more than one population, and were, therefore, considered as evidence of an admixed population. Four individuals from Newfoundland and five from Nova Scotia were found to have mixed ancestry and these individuals were also found to be mixed among the three "provincial" clades in the NJ analysis (Fig. 2.10).
i i AH others Newfoundland ! A
/ i /
m • \
•\ /•
/• • \ /mr • * •ft •*\ Cluster 1 Cluster 2 Nova Scotia New Brunswick
Figure 2.12. A triangle plot displaying the estimated membership coefficient for each individual into its assigned population (Newfoundland, New Brunswick and Nova Scotia), where distance from each of the three corners represents probability of membership (q).
53 AMOVA analyses based on AFLP data showed a moderate level of differentiation among the provinces with high significance (®ST~ 0.203, P< 0.001) where
80% of the variation was found within populations and 20% among populations.
Similarly, an AMOVA based on the mitochondrial COI gene also exhibited a moderate differentiation among provinces (see above mitochondrial DNA sequencing analysis). A separate analysis based on the two mtDNA clades A and B also confirmed that most of the variation was found within populations (98.8%) rather than between. Virtually no genetic divergence was detected between clades (®ST = 0.012) (i.e., between the two putative species as defined by mtDNA). In contrast, the proportion of variation within and among provinces was almost reversed for the COI sequence data with most of the variation being among rather than within populations.
Discussion
Taxonomic implications
(A) Morphology
Morphological characteristics traditionally used to distinguish P. cataracta and P. fragilis were not found to be adequate enough to reliably differentiate the two putative morphospecies in Atlantic Canada. A principle component analysis failed to recognize two separate species {P. fragilis and P. cataracta). That said, many of these descriptions are extremely subjective and vague as has been recognized by previous authors (Hanlon and Smith, 1999; Cyr et al, 2007). In addition, Cyr et a!., (2007) found that a discriminant analysis based on twelve conchological measurements had limited success
54 in differentiating among Pyganodon species. Moreover, the morphological variation that was observed in the Cyr et ah, (2007) study may possibly represent clinal variation in their specimens and not distinct, species-level morphological differences. Conchological characteristics such as colour of periostracum among Atlantic Pyganodon populations seemed to vary with local environmental conditions and/or type of substratum. This observation is anecdotal however, and future studies could focus on examining conchological variation with respect to substrate as these data were not explicitly collected and analyzed in this study. Although minor groupings of individuals were found to cluster together based on province of origin this result is not surprising as individuals from the same lake or pond will look similar based on local environmental conditions. Additionally, beak sculpture was difficult to determine in larger older individuals due to erosion of the umbone but regardless, this characteristic was not consistent as double loop and single loop structures were commonly found on both presumptive species.
Convergence and parallelism of shell shape and external characters are common in freshwater mussels and are thought to have resulted from adaptation to similar environments, such as substrate composition or water velocity (e.g., Johnson, 1970;
Watters, 1994). Similarly, a study by Hanlon and Smith (1999) found that periostracum colour was linked to habitat conditions among P. cataracta in Maine and was therefore, an unreliable characteristic. This study also determined that stomach anatomy was unable to differentiate between these taxa which was in disagreement with a similar study conducted by Kat (1983, 1986) who claimed Pyganodon from Maine closely resemble P.
55 fragilis. Based on the results of the Hanlon and Smith study, P. fragilis is no longer thought to occur in Maine. A closer examination of a larger number external and internal morphological characteristics may reveal more appropriate ways of distinguishing between Pyganodon species, however, given the genetic data presented here this seems unlikely.
(B) Genetic divergence
Although two mtDNA clades were observed, which are consistent with the clades identified by Cry et al. (2007) as putative "P. cataracta" and "P. fragilis", these clades in
Atlantic Canada were shown to exhibit fairly low levels of genetic diversity and extremely low divergence values for both mitochondrial (COI) and nuclear (ITS-1) sequence data. Each "species" was dominated by a single haplotype for the COI gene and most haplotypes differed by only a single base pair substitution. Unique haplotypes did occur but were generally restricted to a single locality. Even though "P. cataracta " was slightly more diverse with two fairly common haplotypes and a larger number of unique haplotypes, it still displayed relatively little genetic variation. This low level of genetic diversity was particularly pronounced among populations attributed to "P. fragilis ", which may be an indication of a recent bottleneck or founder event (Hartl and
Clark, 1997).
Dominant COI haplotypes of both putative species were widely distributed among all provinces and subdrainages with no clear geographical correlation, suggesting a high
56 degree of gene flow throughout Atlantic Canada. Large effective population size and/or high gene flow across large geographic areas are the most probable explanations for the absence of geographical structuring (Waples, 1998; Whitcock and McCauley, 1999).
Populations of Pyganodon are considered secure and widespread throughout Atlantic
Canada and their distribution and abundance has been attributed to their ability to tolerate different environmental conditions and parasitize a wide variety of fish as hosts
(Metcalfe-Smith and Cudmore-Vokey, 2004).
Despite there being little geographic structuring in the COI sequence data, putative "P. fragilis " or Clade A populations did appear to be somewhat more prominent in northern regions of Newfoundland and New Brunswick. For example, only one population assumed to be "P. cataracta " from Clade B was found in southwestern
Newfoundland. Interestingly, only four lakes, rivers or ponds out of 33 surveyed were found to contain both clades A and B in a single body of water. These included Lake
O'Law in Cape Breton, Baker Lake in northwestern New Brunswick, Lake Utopia in southern New Brunswick and Winter River in Prince Edward Island. More extensive surveys may reveal additional lakes, ponds and river comprising both clades.
The distributional patterns of mussels are directly related to the distribution of their fish hosts (Watters, 1992) and the migrational patterns offish hosts may have a large impact on the amount of gene flow occurring within unionid populations (Elderkin et ah, 2007). Potential fish hosts of Pyganodon in Atlantic Canada include the three- spined stickleback, Gasterosteus aculeatus, the white sucker, Castostomus commersoni
57 and the yellow perch, Percaflavescens, all of which are widespread throughout eastern
Canada and are local migrants between lakes and tributaries (Scott and Crossman, 1973).
Therefore, the use of anadromous and/or widespread, vagile fish hosts may increase gene flow among Atlantic Pyganodon populations. The three-spined stickleback, Gasterosteus aculeatus, is widely distributed within the Northern hemisphere and is considered an anadromous and freshwater fish species (Scott and Crossman, 1973). Having the ability to alternate between fresh and salt water habitats allows glochidia the opportunity to colonize new habitats and increase gene flow among relatively distant geographic ranges by exploiting the movements and migration patterns of their host fish (Kat, 1983).
Sequence divergence levels observed for the COI gene (0.5%) are consistent with intraspecific polymorphism seen in other studies of unionids (0-2.82%) whereas typical interspecific values commonly range from 3-6% among unionids (Roe and Lydeard,
1998; Roe et ah, 2001; Machordom et ah, 2003). Similarly, divergence between P. cataracta and P. fragilis for both male and female mtDNA were found by Cyr et al
(2007) to be within this intraspecific range (<1.2%). Likewise, sequence variation between unionid species in the Potalimus and Lasmigona genera ranged from 1.2-15% for the female COI gene (Roe and Lydeard, 1998; King et ah, 1999). Nonetheless, genetic distances among recently diverged taxa are often quite low and would probably reflect the length of time since populations were either reproductively or geographically separated (Nei and Kumar, 2000).
58 Although the AMOVA results for the COI sequence data support the genetic distinctiveness of P. cataracta and P.fragilis these differences were not correlated with any nuclear (AFLP or ITS sequence), geographical or morphological differentiation therefore, there is no concrete evidence to suggest separate species status. Most of the variation observed was among rather than within populations which may reflect historical rather than current processes. These results support a recent and possibly incomplete separation among P. cataracta and P. fragilis (Randy Hoeh, pers. comm.). In fact, it is highly likely that gene flow is occurring between clades where their distributions are found to overlap.
Sequences for the ITS-1 gene were identical for all specimens examined and displayed no genetic variation within or among presumptive P. cataracta and P. fragilis populations, further suggesting that these species are not clearly differentiated. Although not used extensively in unionid phylogenetic or phylogeographic studies, ITS-1 gene sequences have been shown to an effective molecular marker for discriminating among closely related Lasmigona species (King et ah, 1999) but has had limited success in differentiating interspecific differences in Cyprogenia, Epioblasma and Pyganodon species (Jones, 2006; Serb, 2006, Cyr et al., 2007).
Divergence between P. fragilis and P. cataracta was extremely weak for both the mitochondrial (COI) and nuclear (ITS-1) genes and a neighbor-joining and maximum parsimony analysis for both genes formed a single monophyletic group, suggesting the presence of a single species. Although the unrooted phylogenetic trees based on COI
59 gene sequences identified two distinct groups and these groups contained specimens previously identified as P. cataracta from P. fragilis in the GenBank database, there was only extremely slight differentiation between the two clades. Low divergence values in nuclear and mitochondrial sequence data are in agreement with a recent study of
Pyganodon populations in Quebec conducted by Cyr et ah, (2007) who analyzed both male (albeit only six males) and female mitochondrial COI data in addition to the nuclear
ITS-1 gene.
It should be reiterated that specimens were identified as P. cataracta or P. fragilis based on reference samples obtained from GenBank. COI sequences for two P. cataracta and one P. fragilis specimens were from the aforementioned study by Cyr et ah, (2007) and one COI sequence for P. fragilis was obtained from a study conducted by
Hoeh et ah, (1996). No morphological descriptions were provided for these representative specimens therefore, there is no way of knowing what characteristics were used to definitively identify them to species. Given their morphological similarities and difficulties in accurately delineating these putative species it is possible that these specimens were misidentified. This is particularly plausible considering that until recently it was not believed that P. cataracta occurred in Newfoundland. However, this potential complication becomes irrelevant if P. cataracta and P. fragilis are indeed considered conspecific. All available COI sequences on the GenBank database from the
Cyr et ah (2007) study were used as a reference and comparison samples. Interestingly, one of the "cryptic" Pyganodon lineages discovered by Cyr et ah (2007) in Quebec was found to match a COI sequence of P. lacustris from Minnesota donated by Randy Hoeh.
60 The nuclear ITS-1 NJ and MP analysis failed to separate P. cataracta and P. fragilis into two distinct groups and this may be due in part to the slower substitution rate of the noncoding region of rDNA compared to the COI gene (Simon et al., 1994; King et ah, 1999). Incongruent phylogenies between nuclear and mtDNA may represent differences in the evolutionary history of the mitochondrial and nuclear genomes (King et ah, 1999). In most animals, mtDNA inheritance is strictly maternal; however, freshwater mussels exhibit doubly uniparental inheritance or DUI, where there are two mtDNA genomes transmitted through males (the M-type genome) and females (F-type genome)
(Skibinski et al., 1994; Zouros et ah, 1994; Hoeh 1996a). Although male mussels will contain both male and female mitochondrial genomes we only examined the female genome and therefore our COI data only reflects female mediated dispersal, gene flow and patterns of selection. In contrast, AFLP markers are biparentally inherited nuclear markers and would reveal both male and female patterns of dispersal and gene flow.
Furthermore, because the effective population size of the mitochondrial genome is four times smaller than the nuclear genome, due to uniparental transmission of genes, it is more prone to the effects of genetic drift and will have a much faster lineage sorting rate
(Zhang and Hewitt, 2003; Avise, 2004). Hence, the genetic diversity observed when analyzing the nuclear genome will be buffered against the effects of random genetic drift more so than the mitochondrial genome. Difficulties in isolating the male mitochondrial genome prevented us from directly and readily assessing hybridization among these putative species.
61 Another important consideration is that gene trees (e.g., the COI tree) may not accurately reflect the "true" species tree (Hudson 1990, Avise, 2000). Therefore, one must be cautious when estimating species trees from gene trees especially if only a single gene from the mitochondrial genome is used. Many factors such as introgression, lineage sorting and hybridization can affect phylogenetic reconstruction (Sites and Crandall,
2004). Alternatively, low genetic variability may be an indication of a "selective sweep," where a positively selected mutation may become fixed within a population and carry with it other closely linked genes (Avise, 2004). This is especially pertinent when estimating phylogenies and divergence between closely related species. Gene trees derived from multiple independent nuclear markers such as amplified length polymorphisms AFLPs are more likely to represent a "true" species tree (Avise, 2004).
(C) Population genetic structure
In contrast to the sequence data, the AFLP's revealed significant geographical structuring among Pyganodon populations based on STRUCTURE results.
STRUCTURE correctly assigned most individuals to their province of origin uncovering a slightly different pattern of genetic structure compared to the sequence data. Whereas the COI sequence data weakly differentiated populations based on putative species designations and no geographic structuring was observed, the AFLP data showed no clear separation between the presumptive species and moderate geographical structuring.
This pattern of structuring among provinces suggests that current gene flow is occurring more frequently within provinces rather than among provinces (as would be expected)
62 and indeed an AMOVA on both AFLP data and COI sequence data found most of the variation was within (>80%) as opposed to among provinces. Accurate assignments of
individuals to province together with 0ST values ranging from approximately 0.17-0.20
for the AFLP and COI sequence data, suggests moderate genetic differentiation between
Pyganodon populations in Nova Scotia, New Brunswick and Newfoundland.
Nevertheless, with most of the variation being found within rather than among populations suggests that these populations are genetically fairly similar.
The neighbor-joining analysis showed only weak support for separating mussels based on province. The NJ tree was left unrooted and if rooted with an outgroup would form only a single monophyletic group, thus contradicting the notion of any significant
genetic differentiation among provinces. No significant genetic differences were
observed between the putative species based on COI sequence and AFLP data further reinforcing the evidence in support of merging P. cataracta and P. fragilis into a single species rather than two separate species.
Genetic structuring based on geography often follows an isolation by distance pattern, where populations closer together geographically will be more similar genetically
(Kimura and Weiss, 1964; Slatkin and Maddison, 1990). Other unionids studies have also displayed a strong correlation between geographic and genetic distances (Kelly and
Rhymer, 2005) with some species exhibiting this pattern across large (> 1000km)
distances (Berg et ah, 1998; Elderkin et ah, 2007). Furthermore, because freshwater mussels exhibit a patchy distribution with populations scattered unevenly in mussel beds
63 implies that a stepping stone model of dispersal may explain this isolation by distance pattern (Berg et ah, 2007; Zanatta and Murphy, 2007). This is not surprising given that the suspected fish hosts of Pyganodon in Atlantic Canada (the white sucker and yellow perch) migrate locally from lakes to rivers and streams for spawning purposes.
Local fish host migration may aid mussels in colonizing new habitats and increase gene flow among interconnected freshwater systems within a particular province while making dispersal between provinces more challenging, without assistance from an anadromous fish host. The Threespined Stickleback a known fish host of P. cataracta is anadromous and may be contributing to gene flow among these provinces. Although to our knowledge, no published studies have explicitly examined the ability of glochidia to survive in saltwater, some preliminary studies conducted by Mark Gordon (pers. comm.) have indicated that glochidia can survive this transition from freshwater to saltwater and back to freshwater while infecting a suitable fish host.
Due to the presence of admixed populations one might infer that gene flow is occurring between populations in Newfoundland and New Brunswick and between New
Brunswick and Nova Scotia populations. However, given the geographic distance and physical barrier (i.e. the Gulf of St. Lawrence) the evidence for admixture between
Newfoundland and New Brunswick is likely due to retention of shared ancestral polymorphisms. Current gene flow is more likely occurring between New Brunswick and Nova Scotia although it would likely be limited by the relatively narrow size of the
Isthmus of Chignecto.
64 Historical Biogeography
The evidence provided in this study demonstrates that P. cataracta and P. fragilis represent a single species, with the former having nomenclatural priority. P. cataracta, therefore, was found to occur in Newfoundland, Nova Scotia, New Brunswick and Prince
Edward Island and displayed a relatively continuous distribution across all the four provinces. Populations have also been confirmed in eastern Quebec by a recent study conducted by Cyr et al. (2007).
Two possible scenarios could explain the current distribution and post glacial history of Pyganodon populations in Atlantic Canada based on the data presented in this study: either (1) all haplotypes persisted in a single glacial refugium during the last glaciation or (2) the groups of haplotypes representing Clade A and Clade B ("P. fragilis " and "P. cataracta," respectively) arose in two separate refugia and have come into secondary contact throughout various parts of Atlantic Canada. High intraspecific diversity in some species has been proposed to provide evidence for multiple refugia
(Zamudio and Savage, 2003). Due to the low genetic diversity and low variability of haplotypes observed a relatively recent origin for all Atlantic Pyganodon populations is a likely option. Under this scenario you would expect to see a random distribution of haplotypes throughout the colonized provinces (Avise, 1987), however, there is a slight difference in the frequencies of the haplotypes and distribution of haplotypes and this
65 could be due to chance events during dispersal and/or genetic drift following recolonization.
Nonetheless, genetic diversity did seem to be reduced in these northern areas or among clade A and this may once again imply a separate origin in a glacial refugium for this particular clade. For instance, reduced nucleotide diversity with increasing latitude is common among northern fish species, and areas that were previously covered by glaciers usually exhibit lower diversity with only a small number of predominant haplotypes
(Bernatchez and Wilson, 1998). This observation could explain the widespread distribution of only three main haplotypes for all of the Atlantic Pyganodon populations, with 88% of all individuals sampled belonging to one of these three haplotypes.
Increased distance from glacial refugia has also been shown to correspond to a decrease in genetic diversity (Hewitt, 1996; Armbruster et ah, 1998; Freeland et ah, 2004).
The other option may be that the slight difference in haplotype frequencies may indicate the possibility of two separate refugia. This situation may explain the prevalence of Clade A in northern regions of both Newfoundland and New Brunswick and the occurrence Clade B in southern areas. Haplotypes from clade A may have persisted in a northern refugium such as the proposed northeastern coastal or "Acadian" refugium, which was thought to extend from the southeast coast of Newfoundland to Georges banks
(Pielou, 1991; Bernatchez and Dodson, 1991; Bernatchez, 1997) and dispersed south.
The southern clade B may have originated in a more southern Atlantic refugium and is currently dispersing north. This hypothesis agrees with Cyr et ah, (2007) who suggested
66 that a slight discontinuation in the distribution of what they referred to as P. cataracta and P. fragilis in Quebec may indicate that they survived the last glaciation in two different regions (i.e., a southern and northern refugium, respectively). In support of this view, Bernatchez (1998) stated that northern fish species subjected to numerous glacial advances may be have been displaced in distinct refugia by chance events and if separated for only a brief period in evolutionary time may display little or no phylogenetic differences as is observed between the putative "P. cataracta" and
"P.fragilis" mtDNA clades.
Our data do not allow us distinguish between these scenarios. Resolving which of these situations is more likely will require more extensive AFLP or microsatellite research incorporating a greater number of individuals from across the entire range of the species.
Conservation implications
With a high degree of phenotypic plasticity among freshwater mussel species it seems an overreliance of shell characteristics and internal anatomy for classifying unionid taxa may hamper accurate species identifications and further complicate conservation efforts (Kat, 1983, Williams and Mulvey, 1997; Lydeard and Roe, 1998).
This overreliance of shell characteristics and beak sculpture may have led to the initial classification of P. cataracta and P. fragilis as independent species. The results of this study do not support the distinction of P. cataracta and P. fragilis as separate species based on data from both genetic and morphological analyses. Mitochondrial COI and
67 nuclear ITS neighbor-joining and maximum parsimony analyses formed a well supported monophyletic group consisting of both P. cataracta and P. fragilis with minimal if any significant differentiation between these two forms.
Although the AFLP data indicated the presence of three distinct populations based on province, this type of genetic structuring may be an indication of isolation by distance.
This pattern has been shown to occur within unionid species (e.g. Zanatta and Murphy,
2007; Kelly and Rhymer, 2005) and may explain intraspecific geographic structuring among provinces. Conflicting evidence in phylogeographic structuring between mtDNA and AFLPs is presumed by Irwin (2002) to commonly occur in a continuously distributed species with short dispersal abilities. Additionally, recent common ancestry among
Pyganodon populations in Atlantic Canada may explain the discordant geographic structuring seen when comparing sequence data to the AFLP data as has been seen with mtDNA and microsatellites in the northern riffleshell mussel (Zanatta and Murphy,
2007).
Although low sequence divergence values among clades A and B does not support separate species status for P. cataracta and P.fragilis or meet the criteria for designating them as evolutionary significant units, they may represent unique genetic lineages and for purposes of conservation should be considered as discrete management units. Management units reflect current population structure and are defined as populations with significant divergence of mitochondrial or nuclear alleles despite the phylogenetic distinctiveness of those alleles (Moritz, 1994). Additionally, geographic
68 structuring among provinces may warrant recognizing each province as a separate management unit, particularly Newfoundland because it is geographically separated from the mainland populations. Designating these populations a separate management units will allow conservation managers to successfully augment and relocate mussels if populations decline in future and will aid in preserving the genetic integrity of
Pyganodon populations in Atlantic Canada.
Our results are in disagreement with studies conducted by Kat (1983), Kat and
Davis (1984) and Hoeh (1990) who, based on allozyme and morphological data supported recognizing P. fragilis and P. cataracta as distinct species. However, both studies compared P. fragilis from Newfoundland and Nova Scotia to P. cataracta from their southern range in the United States (Delaware, New Jersey and Pennsylvania). It is likely that Pyganodon populations in Atlantic Canada may be genetically different from those found in their southern range due to post glacial history or limitations to dispersal.
Further genetic and morphological studies incorporating P. cataracta from its entire range may shed more light on phylogeographic patterns of this complex.
Although our study was unable to explicitly test for the presence of hybrids, future studies could examine polymorphisms in the male mitochondrial genome and if distinct clades equivalent to the female mitochondrial clades are observed (similar to the
Mytilus edulis and M. trossulus species complex) this could refute our hypothesis of conspecific status for P. cataracta and P. fragilis. Likewise, if the male mitochondrial sequence data formed only a single clade and the male genome in the male individuals
69 was not correlated with specific female mitochondrial clades, this would provide further
evidence to accept our hypothesis of a single species.
In conclusion, we find no evidence to support the designation of P. cataracta and
P. fragilis as two distinct species. Furthermore, P. cataracta and P. fragilis would not fit the criteria of separate species status under either the phylogenetic or genetic species concepts. P. cataracta populations are considered secure and fairly widespread throughout Atlantic Canada and therefore merging these species into a single species would not directly affect its current conservation status. Nonetheless, the results of this research provide a detailed and comprehensive genetic study of P. cataracta in Atlantic
Canada that may prove useful in future conservation studies.
70 Chapter 3. General conclusions and recommendations for the future conservation of freshwater mussels
The systematics of unionoid bivalves has had a problematic history from the level of species to the family level and is continually being reworked as new molecular techniques and analyses become available. The current taxonomy of many freshwater mussel taxa has relied too heavily on morphological characteristics which have been shown to exhibit a high degree of phenotypic plasticity and convergence. Early malacologists described each morphological variant from different geographic locations as unique species which inevitably lead to the over naming or an overestimation in the number of species (Boss, 1971). Subsequent reclassifications have synonymized the names of many species exhibiting this high degree of phenotypic variation into single species but some authors argue that this may have oversimplified the situation among many unionids (e.g., Mulvey et al., 1998; Davis and Mulvey, 1993). Until recently, few phylogenetic studies have been conducted on freshwater mussels in an attempt to rectify these potentially incorrect taxonomies, where named species may not actually represent valid species meeting some rigorous criteria for identifying species (Lydeard and Roe,
1998).
The application of molecular techniques can provide valuable information required to quantify the following: population structure, patterns of variation, gene flow and identification of species as well as contributing to effective conservation strategies of
71 endangered or threatened freshwater mussels (Mulvey et al., 1998). Mitochondrial DNA, with its relatively high rate of mutation, is a powerful marker for reconstructing phylogenetic relationships at or below the family level and mtDNA sequences are particularly useful for solving taxonomic problems (Wan et al., 2004). However, with recent evidence of possible recombination and introgression of mitochondrial genomes
(Tsaousis et al 2005; Shafer and Stewart, 2007) it has become increasingly important to utilize multiple independent nuclear markers to delineate closely related species and infer population history (Zhang and Hewitt, 2003; Wan et al., 2004).
Amplified fragment length polymorphisms (AFLPs) (Vos et al., 1995) are multi- locus markers that can generate hundreds of polymorphic loci to rapidly, efficiently and relatively inexpensively assess genetic diversity and intraspecific variation across the entire nuclear genome of an organism (Mueller and Wolfenbarger, 1999; Bensch and
Akesson, 2005). Despite its obvious advantages as a reliable nuclear marker for systematic and population genetic research few studies have utilized this technique in wild animal populations (Bensch and Akesson, 2005) and fewer still in the study of freshwater mussels (see Mock et al., 2004).
Previous studies based on morphology, stomach anatomy, allozyme and mtDNA have been unable to unequivocally resolve the taxonomic status of the two putative species, P. cataracta and P. fragilis, and many scientists continue to debate their genetic distinctiveness (Clarke and Rick, 1963; Kat and Davis, 1984; Hoeh, 1990; Hanlon and
Smith, 1999; Cyr et al, 2007).
72 In this thesis, the taxonomic status and population genetic structure of P. cataracta and P. fragilis from Nova Scotia, New Brunswick, Newfoundland and Prince Edward Island was examined. By combining both mitochondrial and nuclear sequence data, together with morphology and a powerful multi-locus marker such as AFLPs, this research provides the most comprehensive study to date investigating the genetic diversity and divergence of P. cataracta and P. fragilis in Atlantic Canada.
The results of this study clearly indicate that there is no evidence to the support distinction of P. cataracta and P. fragilis as separate species. All molecular markers employed in this study (AFLP's, COI and ITS genes) support the designation of a single monophyletic group for P. cataracta and P. fragilis. Although two mitochondrial clades are present in Atlantic Canada, consistent with the results of Cyr et ai, (2007), the genetic divergence between these clades is extremely small. Additionally, the two clades are fairly widespread throughout Atlantic Canada and there is no evidence of corresponding nuclear divergence correlated with the observed mtDNA divergence.
Furthermore, a morphological analysis failed to differentiate the putative species based on common morphological characteristics. Therefore, we recommend merging P. cataracta Say, 1817 and P. fragilis Lamarck, 1819 into a single species with the former having nomenclatural priority. Accordingly, the distribution of P. cataracta has now been established with confirmed populations in Nova Scotia, New Brunswick,
Newfoundland and Prince Edward Island.
73 Although preliminary these data also provide insight into the historical
biogeography of P. cataracta and possible colonization routes within Atlantic Canada
after the last glaciation. Additional studies incorporating nuclear markers such as the
AFLPs or microsatellites in conjunction more extensive sampling from the entire range of
this species may provide more conclusive evidence of their phylogeographic history.
Populations of P. cataracta (including populations previously attributed to P. fragilis) are considered secure, fairly common and widespread in Atlantic Canada and
throughout most of its range in the southeastern United States (Williams et ah, 2003).
Their abundance and common distribution has been attributed to their ability to tolerate
varying environmental conditions and their low host specificity (Metcalfe-Smith and
Cudmore-Vokey, 2004). Even though populations of P. cataracta appear relatively
stable, it is, nonetheless, imperative to quantify genetic variation among populations and
determine taxonomic status of this species before populations begin to decline.
Given the current conservation status of freshwater mussels in North America,
accurate systematic relationships and information regarding population genetic structure
is needed for effective conservation management programs. The introduction of exotic
species such as the Asiatic clam, the zebra mussel and the quagga mussel and increasing
anthropogenic pressures on our freshwater ecosystems will inevitably lead to future
extinctions of many more freshwater mussels species. Recent recovery programs of
endangered freshwater mussels have attempted to augment or translocate populations to
preserve genetic variability and viability by increasing population numbers (Lydeard and
74 Roe, 1998; Mulvey et ah, 1998; Serb, 2006). Therefore, accurate assessment of genetic diversity must be incorporated into conservation management practices before freshwater mussels are relocated or reintroduced into new populations. This will maximize the success of augmented populations while avoiding the harmful effects combining genetically distinct populations leading to outbreeding depression (Lydeard and Roe,
1998; Mulvey et ah, 1998). For example, if populations from Newfoundland were declining, Pyganodon mussels with similar nuclear and mitochondrial profiles from the mainland should be used to relocate or reintroduce populations into Newfoundland.
Future studies of P. cataracta should concentrate on more extensive surveys of this species incorporating multi-locus nuclear markers such as AFLPs or microsatellites to further investigate their population genetic structure and historical biogeography throughout their North American range. Additionally, more comprehensive study of potential fish hosts could provide insight into their genetic structure and distribution and could greatly aid conservation efforts if needed in future.
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