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5-1994
Mitochondrial DNA Variation Within the Western-Clark's Grebe Complex (Podicipedidae: Aechmophorus)
Bruce Alan Eichhorst
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Recommended Citation Eichhorst, Bruce Alan, "Mitochondrial DNA Variation Within the Western-Clark's Grebe Complex (Podicipedidae: Aechmophorus)" (1994). Theses and Dissertations. 3349. https://commons.und.edu/theses/3349
This Dissertation is brought to you for free and open access by the Theses, Dissertations, and Senior Projects at UND Scholarly Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of UND Scholarly Commons. For more information, please contact [email protected]. MITOCHONDRIAL DNA VARIATION WITHIN' THE WESTERN-CLARK" S GREBE COMPLEX ( POOICIPBDIDAE :. ABCHMOPHORUS')
by
Bruce Alan Blchhor.st Bachelor of Science, Unive:rsi.t.y of Wisconsin-Oshkosh, 1982 Master of Sclence, Universi:ty of: Wisoonsin.-Oshkosh, 1986
A Di.ssertation. Submit.tad to the Graduate Faculty o.f the Uu:i vers.i'ty of North Dakota .ir. partlal. fulfillment, of 'the requirements for the de.gr.·e.e of
Doct.<)r of: Philosophy
Grand Forks, North Dakota May 1994 Copyrighted by Bruce Alan Eichhorst 1994
ii This dissertation, submitted by Bruce Alan Eichhorst in partial fulfillment of the x:equirements for the Degree of Doctor of Philosophy from the Univers.ity of North Dakota, has been read by the Faculty Advisory Committee under whom the work has been done and is hereby approved ..
This dissertation meets the standards for appearance, conforms to the style and format requir·ements of t.he Gradua.te School of the Univer·sity of Mort.h Dakota, and is hereby approved ..
_fl~ Dean of the G1du~ Scho~l t*~ i. ,,,,
iii PERMISSION
Title Mitochondrial DMA Variation Within the Western-Clark's Grebe Complex (Podicipedidae: Aechmophorus) Deportment Biology Degree Doctor of Philosophy
In presenting this dissertation in partial fulfillment of the requirements for a graduate degree from the University of North Dakota, I agree that the library of this University shall make it freely available for inspection. I further agree that permission for extensive copying for scholarly purposes may be granted by the professor who supervised my dissertation work or, in his absence, by the chairperson of the department or the dean of the Graduate School. It is understood that any copying or publication or other use of this dissertation or part thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given me and to the University of North Dakota in any scholarly use which may be made of any material in my dissertation ..
Signature ~(_4,~ Date ,;>. 7 ~ /7"71
iv TABLE OF CONTENTS
LIST OF ILLUSTRATIONS ...... vii
LIST OF TABLES., ...... ix
ACKNOWLEDGME1'iTS ...... xi
ABSTAACT ••••••••••• " ..... ~ ...... xiii CHAPTER
I • GENERAL INTRODUCTION ...... 1
II. BACKGROUND ON THE GENUS AECHMOPHORUS .••••.•..••..••• 4
Nomenclatural History ...... 4 Fossil Histo·ry ••••••••••••• ,. ••••••••••.••••••••. 11 Current Distribution and Abundance.~·······~·~·-16 Migratory Movements •••••.••••••••••••••••...••••• 22
III. MITOCHONDRIAL DNA DIFFERENTIATION, POLYMORPHISM, AND POPULATION STRUCTURE IN THE WESTERN-CLARK'S GREBE COMPLEX (AECHMOPHORUS) OF THE UNITED STATES AND CANADA ...... 2 6
Introduction •.••••..•...••.••.••••..••.••••••••• 26
Materials and M~thods •••••• $ •••••••••••••••••••• 30 Choice of Sample Populations and Field Collections •••••••••••••••••••••••••••••••••• 30 Laboratory Analysis ••.•••••••.•.••••.••••••.• 34 Data .Analysis ...... 35 Results ...... , •••••••••.••••• • 42 Mitochondrial-DNA Genome •••••••••••••••••••.• 42 Haplotype Phylogeny and Diversity .•••••.•.••• 44 Nucleotide Diversity and Divergence •..•••.••• 51 Population Genetic Variation ••.....•.. ~ •.•.•. 55 Discussion ••••.•••••.•..••.•.•••...••••.•••••••• 58 Phylogenetic Status of the Color Phases .••••• 58 Genetic Divergence Between the Color Phases,, .. 63 Intraspecific Phylogeography and Female mediated Gene Flow in Aechmophorus ..•.••••••• 68 Long-term Effective Population Size of Aechmophoz:us Females ...... 7 0 Evolutionary and Taxonomic Implications •.•••• 72
V IV.. A REVIEW OF SPECIATION MODELS PROPOSED FOR THE WESTERN-CLARK'S GREBE COMPLEX (AECHMOPHORUS) •••.... 75 Introduction ••••..•••.•••.••..•••••••.••••••..•• 75 Quaternary History of Western North America •••.• 76 Aechmophorus Distribution and Migration ••••••••• 81 Aechmophorus Speciation Models •••••••••••••••••• 82 Discussion •.••••••••••••••••••••••••• o••••••••••84 Ratti's Models ••••• t••··~····················84 Feerer's Model ••••••••• ~·····················87 Conclusions •••.•.••••••••••••••••••••••••••••••• 88
V. RECOMMENDATIONS FOR FUTURE RESEARCH ON THE EVOLUTIONARY HISTORY OF THE AECHMOPBORUS GREBES •••• 89
APPENDIX ...... 9 3
LITERATURE CITED ...... 9 5
vi 1.. tocati OJfits wheilt.'e Ae:
vii Figure Page
5. Unrooted parsimony network summar.1.z.1.ng restriction site changes between the 12 Aechmophorus m'LO-NA haplotypes. {a) Fragment patterns [A-CJ produced by the enzymes listed. Direction of arrow indicates inferred restriction site loss between adjacent fragment patterns. (b) Most parsimonious, unrooted network produced by both Dolle and Wagner parsimony methods, with the site changes indicated between adjacent haplotypes •••••• 38
6. UPGMA phenogram of the 12 Aechmophorus mtDNA haplotypes, based on genetic distances (d) between haplotypes ••••••••••••••••••••••••••••• 50
7. Map showing extent of Late Wisconsin glaciers in the Cascaae and Sierra Nevada ranges, and pluvial lakes within the Great Basin, western United States. Map was compiled and modified from maps in Porter et al. (1983) and Williams and Bedinger (1984)···~····························80
viii LIST OF TABLES Table Page
1. Aechmophorus fossil localities and the estimated ages of the fossil-bearing deposits. Distribution of localities shown in Figure 1 {identified by No.) ••••••••••••• ~··················14 2. Relative frequencies(%) of Clark's Grebes in various Aechmophorus breeding populations •••••••••• 19
3. Frequencies of mtDNA fragment patterns produced by restriction enzymes whose restriction sites were polymorphic within the total sample of 109 Aechmophorus grebes •••••••••••• 45
4. Distribution of rntDNA haplotypes within the Aechmophorus :;;,henotypes ...... 46
5. Estimates of number of nucleotide substitutions per site (d) between 12 mtDNA haplotypes observed in 109 Aechmophorus grebes. d-values are above the diagonal, standard errors below the diagonal. All values expressed as percent. See Table 4 for genotypes ...... 4 7
6. Geographic distribution of Aechmophorus mtDNA hap 1 o type s • • • • • • • • • • • • • • • • • • • • • • • • • • .. • • • • • • • • • • • • • • 4 8
7. Estimates of mtDNA nucleotide (rr) and haplotypic (h) diversity in the Aechmophorus grebes surveyed in this study. rr expressed as percent ••...•••.••••••••••••••••••••••••••••••••••• 52
8. Average number of mtDNA nucleotide substitutions per site (dXY) and average number of net nucleotide substitutions per site(~) between Aechmophorus phenotypt.~s, and between geographic regions. All values expressed as percent ...... 5 3
ix Table Page
9. Average number of nucleotide substitutions per site ( dxn above diagonal; SE below diagonal) between Aechmophorus breeding sites. All values expressed as percent •••••••••••••••••••••••• 56
10. Average number of net nucleotide substitutions per site ( dAJ between breedj ,1g sites. All values expressed as percent •••••••••••••••••••••••• 57 11. Intrademe identity probabilities (underlined) for Aechmophorus breeding sites, and interdeme identity probabilities (below diagonal) between breeding sites ••••.•••• * ••••••••••••••••••••••••••• 58
12. Hierarchial analysis of molecular variance (AMOVA) on two matrices of mtDNA genetic distances between Aechmophorus haplotypes (see Materials and Methods for explanation). Only SW (breeding sites EAGL, GOOL, UKLL) and NE (ALKL, DELM, LOSA) geographic regions used in analysis •••••.••••••••••.•••••••••••••••••••••••••• 59
13. Estimated mtDNA genetic distances (percent nucleotide sequence divergence) reported for pairs of closely related North American avian taxa. Since few of the below sources calculated net divergence (dA) all percentages listed are estimates of average divergence ( dxy) • • •••••••••••••••••••••• ,., ...... 6 0
14. Estimates of mtDNA polymorphism within North American avian species. Some values not provided in original sources are from Table 3 of Avise and Ball ( 1991) ...... 64
15. Comparison of genetic divergence values reported for pairs of North American avian taxa. DNA-hybridization v~lues (delta TM) for Parus taxa are from Sheldon et al. (1992). Delta TM value for Aechmoohorus taxa derived from T 50H value ( using equ... ation 1 of Sheldon et al. 1992) reported by Ahlquist et al. (1987). gcnDNA estimates based on 1:1.7 relationship between delta TM and percent nucleotide divergence (Caccone et al. 1988). Parus mtDNA divergence estimates {from inferred restriction site data) are from Gill and Slikas (1992) and Gill et al. (1993) •..•.....•• 67
X ACKNOWLEDGMENTS First and foremost, I give special thanks to my wife Jean for her constant love, support, encouragement, and patience during my tenure in graduate school. Her understanding of my annual pilgrimages to conunune with the
"grebes 0 is especially appreciated. I wish to thank Dr .. Rob Fleischer, Dr. Rich Crawford, Dr. Al Fivizzani, Dr. Bill Wrenn, and Dr. Jim Waller for serving on my graduate committee. Their patience during the hectic final stage of preparing this dissertation is much appreciated. My advisor Dr., Fleischer graciously provided me with the opportunity to work in his lab, and allowed me the freedom to do my own thing. During Dr. Fleischer's absence from campus, Dr .. Crawford kindly served as my co advisor. My fellow lab-mates Cheryl Tarr, Yvette Matthews, and Liz Rave made the long hours in the lab interesting. During my first summer of collecting grebes, Barry Parkin volunteered to go-along-for-the-ride, and turned out to be a great assistant; thanks Barry. Mike Anderson kindly allowed Barry and I to camp-out and use the facilities at the Delta Waterfowl and Research Statlon while we collected grebes on the Delta Marsh. Jay Bogiatto granted permission to stay at the Eagle Lake Field Station, and made the
xi station's facilities readily available to me,. The gang at the station - Jay, Rhy, Robert, and Teri made my stay there. most enjoyableK Dr ... Bob Storer and Dr. Gary Huechterlein, f ellc,w grebe enthusidsts, have generously sha.red their knowledge about We·stern and Clark Ks grebes wi.tb me over the, yean;.. In exchange, :for all my dead grebes, Dr. Bob Storer gen.::rously provi.ded me with some extr·a ca.sh. •.
Pun.ding for my .research was provided by the Department of Biology's Acade:mic .t?.rogr-ams and Student Awards Committee and the Graduate School ( Summer Doctoral Resea:r·ch Fellowship and Graduate School Research Grant) of the University of
North Dakota; the Frank M. Chaptnan Memor·ial Fund of -the American Museum of Natural His·tory; Sigma Xi; and grants to
Dr. Fleischer from NSF and NSF' s E:PSCoR program.
xii 1!o the memory of my mother, Mildred Magdalene Eichhorst, who always encou.raged and supported my endeavor's in life. ABSTRACT
The Western Grebe (Aechmophorus occidenta:lis) and
Clark's Grebe (A .. clarkii) are considered to be closely related .. Until recently, they were regarded as color phases of one species--the Western Grebe. I assessed mitochondrial DNA (mtDNA) variation within and between the Aechmophorus color phases. I also evaluated the plausibility of two vicariance models that have been. proposed to explain the
evolution of the color phases.
Grebes were collected from seven breeding sites,
representing three geographic regions of the United States
Canada breeding range. Restriction-enzyme analysis of a
total sample of 109 grebes was conducted. Estimates of
nucleotide divergence ( dxu dA), nucleotide diversity, and
haplotypic diversity were based on restriction-site data
inferred from observed restriction-fragment patterns.
Population genetic structure was assessed using interdeme
genetic variation ( G1n) and analysis of molecular variance (AMOVA) methods.
MtDNA's of the Aechmophorus grebes showed size
variation and individual heteroplasmy. No fixed fragment
pattern differences were observed between the color phases,
and estimated mtDNA divergence between them was low ( dxy =
xiii 0 .. 19%; dA =zero). The level of nucleotide diversity estimated for the entire sample was 0.18%. Twelve mtDNA haplotypes were observed, with the most common haplotype being represented in 49.5% of the grebes surveyed.
Haplotype frequencies were homogenous among the breeding sites and among the geographic regions. Genetic population structuring was not detected by either the G5 x or the AMOVA method. A polyphyletic pattern in terms of mtDNA genealogy was observed between the color phases. The evolutionary effective population size ( Nr,a,) of Aechmophorus females was estimated to be 17,800. Relative to the current female populaticn size ( about 60,000 to 100,000 females) , the Nft•J estimate suggests that the number of females has been relati '~ly stable over the recent evolutionary past. Of the two vicariance models that I evaluated from a biogeographic perspective, I conclude that the model proposed by John Ratti is more unlikely than the model proposed by Jeffrey Feerer. While the mtDNA results provide only equivocal support of the proposed timing of the vicariance event hypothesized in Feerer's model, conversely the results clearly do not provide evidence for rejection of
Feerer's model either.
xiv CHAPTER I
GENERAL INTRODUCTION
Within the last two decades, advances in DNA technology and the rapid growth of the field of molecular genetics have for the first time allowed evolutionary biologists to directly assess genetic variation within and between species, rather than inferring genetic variation from patterns of morphological variation. The ability to compare
DNA sequences (either directly or indirectly) has resulted in the use of molecular markers appropriate for investigating the recent evolutionary history of newly formed, or incipient species (Harrison 1991).
Studies of mitochondrial DNA (mtDNA) variation in birds have demonstrated the usefulness of using this maternally transmitted molecule for tracing phylogenetic relationships intraspecifically, and interspecifically where avian taxa are closely related (reviewed in Avise and Ball 1991). At the intraspecific level, restriction-enzyme assays of mtDNA variation have revealed a variety of phylogeographic patterns; from species in which a single array of closely related mtDNA genotypes is geographically widespread (e.g.,
Red-winged Blackbird [Agelaius phoeniceus], Ball et al.
1988; Downy Woodpecker [Picoides pubescens], Ball and Avise
1 2
1992), to species in which multiple, genetically-divergent arrays of genotypes are associated with separate geographic regions within the range of the species (e.g., Seaside
Sparrow [Ammodramus maritimus], Avise and Nelson 1989; Fox
Sparrow [Passerella iliaca], Zink 1991). The phylogeographic pattern observed within a particular species provides considerable insight into the recent evolutionary history of the species.
At the interspecific level, fixed mtDNA restriction fragment/site differences have been found between several pairs of closely related avian species (e.g., Mack et al.
1986, Avise and Zink 1988, Zink et al. 1991b, G1ll et al.
1993.), thus raising the hope that the mtDNA molecule can provide a source of genetic markers for distinguishing between othe·r closely related avian species (Avise and Ball
1991).
The Western and Clark's grebes (Aechmophorus occidentalis and A. clarkii, respectively) are considered to be closely related as evidenced by the degree of morphological (Storer and Nuechterlein 1985), morphometric
(Livezey and Storer 1992), and behavioral (Nuechterlein
1981a) similarities between them. These grebes were originally described as separate species in 1858 (in Baird
1858:895), but from 1886 until 1985 they were considered color phases (or morphs) of one species--the Western Grebe.
In 1985, the Clark's Grebe was restored to species rank (AOU 3
1985). This change was prompted by the observation of high
(>95%) positive assortative mating (Storer 1965, Ratti 1979,
Nuechterlein 1981a, Lindvall and Low 1982) and the discovery of a behavioral isolating mechanism (Nuechterlein 1981a).
Two vicariance models have been proposed to explain how, and when, A. clarkii and A. ocaidentalis evolved. One model postulates an episode of isolation and allopatric differentiation during a Pleistocene glacial period (Ratti
1977), while the other model postulates a more recent vicariance event, sometime during the Holocene interglacial period (Feerer 1977).
In the hopes of providing a clearer picture of the evolutionary history of the Aechmophorus grebes, I first assessed mtDNA variation within and between A. clarkii and
A. occidentalis; and second, I evaluated the vicariance models proposed by Ratti (1977) and Feerer (1977).
In Chapter II, I provide the reader with background information on the Aechmophorus grebes. This chapter is not meant to be a complete literature review on Aechmophorus grebes, and some of the information presented is new. In
Chapter III, I present the results of the mitochondrial DNA analysis. In Chapter IV, I review and evaluate vicariance models proposed by Ratti (1977) and Feerer (1977). In
Chapter V, recommendations for future research on the evolutionary history of the Aechmophorus grebes are given. CHAPTER II
BACKGROUND ON THE GENUS AECHHOPHORUS
Nomenclatural History
In 1858, George N. Lawrence (in Baird 1858) described two new species of grebes from 10 specimens collected in the western United States and Mexico. He named them Podiceps occidentalis, Western Grebe, and P. clarkii, Clark's Grebe.
Based on differences in body size, bill shape, bill color and lore color, Lawrence described P. clarkii as "a near ally of 'P. occidentalis,' but I think quite distinct" (in
Baird 1858:895). He assumed that all the specimens were in winter plumage since they lacked the breeding plumage ruffs and crests characteristic of P. cristat;us (type for
Podiceps).
Coues (1862a) erected a new genus, Aechmophorus, for
occidentalis and clarkii, and described a female specimen of clarkii in full breeding plumage that had, "a decided occipital crest" but "no decided colored ruffs" (Coues
1862b:404}. As more specimens became available to Coues, the distinction between the two forms in body shape and bill
shape diminished. Apparently the remaining differences, bill color and lore color, were insufficient characters for
4 5 retaining specific rank, because by 1873, he was treating the two forms as varieties of occiden~alis (Coues 1874).
Henshaw (1881) agreed with Coues, hut others, including
Ridgeway (1881) and Fannin (1891), agreed with Lawrence's classification. The American Ornithologists' Union (AOU) upheld the views of Cones and Henshaw when they published the first Check-list of North American birds (AOU 1886).
Subsequent editions of th~ check-list upheld the decision to call the two forms varieties.
The existence of the two forms was largely ignored until Storer (1965:59) redescribed them ("dark phase"= A. occidentalis, "light phase"= A. clarkii): "there are dark phase birds in which the bill is rather dull greenish-yellow and the black of the crown extends bel0w the eyes, the lores, and the narrow line of bare skin extending from the eye to the gape; and there are light-phase birds which have orange-yellow bills and light faces--the black of the crown not reaching the lores or the eyes." Shortly before
Starer's description, Dickerman (1963~66) found that the resident population of the central poTtion of the Mexican
Plateau was made up of 0 srnall, pale individuals, racially distinct from the breeding populations of the United States and Canada." He separated these small individuals as a subspecies for which he used the name A. o. clarkii. He named the northern subspecies A. o. occidentalis. Dickerman
(1973) subsequently obtained additional Mexican specimens 6 which confirmed that there was little or no overlap in measurements of wing cord or culmen between the subspecies, but that both color phases were present in the Mexican populations, thereby nullifying the "pale" color character he originally reported (Dickerman 1963) as being diagnostic for A. o. clarkii.
Storer {1965) reported high levels of positive assortative mating in a mixed color-phase population at the
Bear River Migratory Bird Refuge, Utah. Equally high levels of assortative mating were later observed at this same refuge by Lindvall (1976). This led Ratti (1977) to undertake a study of the color phases. Ratti also found high levels of assortative mating at Bear River and at various locations in Oregon and California. Ratti's data on spatial distribution indicated that light-phase birds are non-randomly distributed among and within winter and summer flocks, and among and within nesting colonies. Ratti (1979) concluded, based on his data from Utah and California, that dark- and light-phase Western Grebes behave as sep~rate biological species. Thus, he recommended resurrecticn of A. clarkii (light-phase) as a separate species.
Feerer (1977) censused nesting colonies at various locations in Mexico, the United States and central Canada.
He found that a significant proportion of all Western Grebes seen in Mexico were "intermediate plumage" forms (see Feerer
1977:9-11 for definition of plumage forms). In contrast, 7 colonies in the United States and central Canada rarely contained intermediate forms. Light-phase birds were rarely found in central Canada, but were relatively common in
Mexico and California* In addition, Feerer found that the frequency at which each form occurred at nesting colonies was correlated with mean air temperature, latitude and longitude. This prompted him to suggest that the two color phases have different climatic preferences.
Feerer {1977) also studied the foraging behavior of the two color phases and found significant differences between them in time spent foraging, prey capturing ability and prey size utilization. He concluded that his "data strongly support the hypothesis that the two forms of Western Grebe exhibit niche partitioning of food resources in spring"
(Feerer 1977:iv). Nuechterlein (1981a, b, Nuechterlein and Storer 1982) studied the courtship behavior of sympatric populations near the Oregon-California border and allopatric populations of dark-phase birds in Manitoba. Film analysis of the many visual courtship displays revealed no difference between the two color phases in either form, duration, or sequence of the displays. The only difference Nuechterlein observed was in the "Advertising" call, which is used to attract potential mates prior to pair formation~ Dark-phase birds have calls with two notes ("cree-creet"), while light-phase birds have a one-noted call ("creeetn). This discovery 8 prompted Nuechterlein to conduct playback experiments which showed that in the sympatric populations, courting males of both color phases readily distinguished the two call-types and approached or answered female caL.s of their own phase .. But in the dark-phase allopatric populations, males showed very poor discrimination between the two call types .. Nuechterlein also reported that the color phases appeared to feed at different water depths. This was initially based on his film analysis of different dive-types (Nuec1'-t'3rlein 1981a) and later confirmed by observations at feeding sites where water depths had been measured (Nuechterlein and Buitron 1989). The AOU check-list (1983:10) noted for the first time that "two 'color' morphs .... exist in the populations of A. occidenta.lis," and suggested that they may represent "distinct species." Storer and Nuechterlein (1985) conducted an analysis of plumage and morphological characters of the two color phases. They found considerable overlap between the color phases in all plumage color characteristics except facial patterns, and suggested that they should be recognized as separate species if and when the color phases in Mexico are found to be reproductively isolated from each other. In spite of Storer and Nuechterlein's (1985) recommendation, the AOU's Committee on Classification and Nomenclature declared the two color phases distinct 9 biological species, and restored A., clarkii Lawrence (AOU
1985:680-681). The committee's decision was bas.ed on the ecological (Ratti 1979) and behavioral (Nu.echterlein 1981a, Nuechterlein and Storer 1982) studies mentioned above. This change prompted Dickennan (1986) to name the northern light phase population A. c .. transitionalis and the Mexican dark phase population A .. o .. ephemera.lis. Feerer (1977:68} suggested that "genetic analysis of Western Grebe families may provide the essential clue to systematic relationships" and i'further study is needed of the magnitude of gene exchange ...... before the two Western
Grebe forms are elevated to species status.•* Ratti ( 1977} conducted a preliminary survey to determine if any protein variation existed between the two color phase,s. His electrophoretic analysis revealed no differences between the phases, but only general protein and six enzymes were assessed.
Ahlquist et al. (1987) used the technique of DNA-DNA hybridization to measure the divergence between the single copy nuclear DNA ( scnDNA) sequences of A. occidentali.s and A. clarkii. In each of three experimental sets they hybridized radio-labeled scnDNA tracers of each species with driver DNAs of other species. Using the ~50H statistic as an index to the median thermal stability of each DNA-DNA hybrid (Sibley and .Ahlquist 1983), they found that DNA-DNA hybrids between occidentalis and clarkii dissociated at a 10 me:an temperature O. 57{T')C below the melting temperature of int:raspecif .i.c ID']iJA-DllA hybrids, and that this difference was
.h ig. hl y s1gn1.. . f icant.,, Ahlquist et al. (1987:4) concluded that
11 11his level o:f genetic differentiation between. A. occ.identalis and A. clarkii does not bear upon the question of their status tJs biological species.. Ho·wever ,. coupled with their widespread sy1npa1,t:ry, pos.itive assortative mating, and weak morphological differantiation, i.t supports the hypothesis that they are s.epara.te species. 0 Subsequently, Bledsoe and Sheldon (1989a) reanalyzed the Aechmopho.rus data (which by then consisted of ei.ght total experimental sets all of' which had been run on the same thermal chromatography machine), using delta mode as an index to DNA disslmila.r.tt.y. They r'eported a mean modal difference between the two specie:s of 0.82°C. For their comparisons between individuals of the two specie·s, 69% of the heterospecific comparisons had a significantly lower mean mode than the homoduplexes: whereas only 25% of the comparisons between individuals within each species showed a significant reduction i.:n mode. Based on these differences they suggested that there is higher interspecific, than intraspecific, sequence variation in the scnDNA of ilechmopho.n1s.
rrhe dtlta from these eight experiments were subsequently analyzed by V. M. Sarich (pers. comm.), who concluded that the above reported differences between occidentalis and 11 clarkii appeared to be entirely artifactual, apparently due to what he termed a "position effect" (i.e., the w--1.gnitude of the melting point temperature for each DNA-DNA hybrid was partially dependent on where the sample tube had been placed in the thermal chromatography block). Thus, contrary to Bledsoe and Sheldon {1989a), Sarich concluded that the level of intraspecific variation was actually nearly as great as the level of interspecific variation, therefore there was no measurable DNA-DNA hybridiz.ation difference between A. occidentalis and A. clarkii.
F'ossil History
Fossils of Aechmophorus have been found at no less than
18 sites in North America, all west of longitude 98°W (Fig.
1, 'l1able 1) . Two congeners of Recent Aechmophorus have been described, A. elasson by Murray (1967), from the Upper
Pliocene (Hagerman) Glens Ferry Formation of Idaho, and A. lucasi by Miller (1911), from the Late-Pleistocene deposits of Fossil Lake, Oregon. A. lucasi was later considered to represent a chronoclinal subspecies, A. occidencalis lucasi, by Howard (1946), being larger than Recent Aechmophorus.
Most recently, Storer (1989) showed that the Oregon fossils are intermediate in size between the bones of Recent
Aechmophorus from northern (Alberta) and southern
(California) populations, and therefore the recognition of
0 1 ucasi tt as a distinct form of Aechmophorus is not warrnnted. 12
Figure 1. Locations where Aechmophorus fossils have been discovered. Numbers correspond to localities listed in
Table 1. 13
\ I
\ ....) -L_....____ _ 14
Table 1. Aechmophorus fossil localities and the estimated ages of the fossil-bearing deposits. Distribution of localities shown in Figure 1 (identified by No.).
Locality (No.) Location Age ( sources•)
Glenns Ferry Twin Falls Co., Late Pliocene (16) formation (1) Idaho 3.7-3.2 my BP
San Diego San Diego Co. , Late Pliocene (4) formation (2) California 3 . 0-1. 8 my BP
Lake Chapala (3) Jalisco, Mexico Pleistocene (2,20)
Chirnalhuacan (4) D. F., Mexico Late Pleistocene (3)
Fossil Lake (5) Lake Co., Oregon Pleistocene (1,10)
Connley Caves Lake Co. 1 Oregon Holocene (7) ( 5) 9,500-7,200 yr BP
Burnet Cave Eddy Co., Late Pleistocene (9, ( 6) New Mexico 19)
Lake Manix San Bernardino Pleistocene (12,13,15) ( 7) Co., California 300,000-20,000 yr BP
China Lake Kern Co., Late Pleistocene (6) ( 8) California > 19,000 yr BP
Kokoweef San Bernardino Late Wisconsin (14,17) Cave ( 9) Co., California
Rodeo Station Alameda Co. , Pleistocene (15) ( 10) California
Chandler Sand Los Angeles Co., Sangamon (14) Pit ( 11) California 130,000-120,000 yr BP
San Pedro Los Angeles Co., Sangamon (14,15) Lumber Yard ( 12) California 130,000-120,000 yr BP
Del Rey Hills Los Angeles Co., Sangamon (14,15) ( 13) California 130,000-120,000 yr BP
Newport Bay Orange Co., Sangamon (11,14) Mesa ( 14) California 130,000-120,000 yr BP 15 Table 1. Continued.
Locality {No.) Location Age ( sources")
San Miguel California Late Pleistocene (8) Island (15) 39,000-25,000 yr BP
Charlie Lake British Columbia Holocene (5) Cave (16) 10,000-0 yr BP
Bear River Box Elder Co., Holocene (19) ( 1 7) Utah 1,000-760 yr BP
4 (1) Allison 1966; (2) Alvarez 1977; 3) Brodkorb and Phillips 1973; {4) Chandler 1990; (5) Driver 1988; (6) Fortsch 1978; (7) Grayson 1979; (8) Guthrie 1992; (9) Hester 1967; (10) Howard 1946; (11) Howard 1949; (12} Howard 1955; (13) Jefferson 1985; (14) Jefferson 1991; {15) Miller and DeMay 1942; (16) Murray 1967; (17) Norell 1986; (18) Parmalee 1980; (19) Schultz and Howard 1935; (20) Storer 1992.
Several deposits of Pleistocene age have yielded
Aechmophorus fossils (Table 1). Deposits from Fossil Lake,
Oregon have yielded the greatest number of bones, more than
800 {Howard 1946). The Fossil Lake basin was occupied by a large, deep lake (Fort Rock Lake) during the Pleistocene, which probably had dried up completely by 8,000 to 9,000 years ago (Allison 1966). This basin was one of more than a hundred hydrologically closed basins in the western United
States and Mexico containing "pluvial lakes" during the
Wisconsin glaciation (Bradbury 1971, Smith and Street-Perrot
1983, Benson and Thompson 1987, Forester 1987). Radiocarbon dating of deposits at both Fossil Lake and nearby Connley
Caves (also located in Fort Rock Lake basin), indicates that 16
Aechmophorus grebes were present at Fort Rock Lake at least as far back in time as 29,000 years ago (Allison 1966) and probably utilized the lake until it dried-up (Grayson 1979).
The presence of Aechmophorus fossils at other pluvial
lake sites (e.g., China Lake, Lake Manix, Kokoweef Cave,
Burnet Cave, Chimalhuacan, Lake Chapala; see Fig. 1 and
Table 1) strongly suggests that Aechmophorus grebes likely
utilized many of the pluvial lakes that were present during
the Wisconsin glaciation.
Current Distribution and Abundance
During the breeding season, Aechmophorus grebes require
fresh water lakes/marshes which have extensive areas of open
water bordered by emergent vegetation, and which support
large populations of fish (Nuechterlein 1975). Nests are
built over water, typically in dense colonies. Colony
locations within individual lakes/marshes can change from
year to year depending on water conditions (Nuechterlein
1975, Eichhorst pers. observ.). Entire lake/marsh systems
may also become unsuitable for nesting during periods of
drought or when water levels are changed appreciably by
human activity (Eichhorst and Parkin 1991, Eichhorst pers.
observ.).
Western and Clark's grebes are syrnpatric throughout
much of their North American breeding range (Fig. 2), with
the relative frequency of A. clarkii generally declining
with increasing latitude (Table 2). Aechmophorus 17
Figure 2. Distribution of Aechmophorus grebes during the breeding season (stippled areas). Breeding occurs at widely scattered suitable lakes/marshes within these areas.
Number shown for each state/province is a rough estimate of the yearly breeding population sizeo See Current
Distribution and Abundance for details of these estimates. 18
Kilometers 19
Table 2~ Relative frequencies{%) of Clark's Grebes in various Aechmophorus breeding populations.
Country State/Province County Location Year % Sourcea
Mexico Northern and ? 100 2 central states 1976 83 5
Western and ? 31 2 southern states 1976 67 5 United States Arizona Mohave Havasu NWR 1990 83 8 California Mono Topaz 1976 21 5 Lake Clear Lake 1976 60 5 Lassen Eagle Lake 1976 13 5 1977 2 12 1990 18 3 Modoc Goose Lake 1977 95 12 1990 >90 3 Siskiyou Tule Lake NWR 1976 20 5 1977 13 12 Lower Klamath 1976 20 5 NWR 1977 21 12 Del Norte Lake Earl 1976 3 5 Oregon Klamath Upper Klamath 1977 49 12 Lake 1981 38 12 1990 58 3 Utah Box Elder Bear River 1963 12 14 MBR 1974 13 9 1975 18 11 1976 17 5 1977 25 12 Wyoming Statewide survey of seven sites 1990 30 13 North Dakota Kidder Alkaline Lake 1991 10 3 Minnesota Todd Lake Osakis 1989 0 3 Canada Manitoba Delta Marsh 1976 0 5 1989 2 4 Pelican Lake 1986 1 1 20
Table 2. Continued.
Country State/Province County Location Year % Source"
Canada Saskatchewan Old Wives Lake 1957 1 10 1976 1 5 Last Mt. Lake 1989 1 7 British Columbia Duck Lake 1981-3 1 6
4 (1) De Smet 1987; (2) Dickerman 1973; (3) Eichhorst unpubl.; (4) Eichhorst and Parkin 1991; (5} Feerer 1977; (6) Forbes 1988; (7) Gollop 1989; (8) Havasu NWR unpubl. memo; (9) Lindvall and Low 1982; (10) Palmer 1962; (11) Ratti 1977; (12) Ratti 1981; (13) Ritter and Cerovski 1990; (14) Storer 1965.
populations in Mexico are presumably residential, and a
partial count by Williams (1982) of 740 to 840 individuals
suggests that the total Mexican population may consist of
only a few thousand individuals.
Estimates of the total population within the United
States-Canada portion of the range have not been made.
Estimates of population size for each of the states and
provinces within the breeding range are given in Figure 2.
I derived these estimates using distribution and abundance
information from both published (e.g., Koonz and Rakowski
1985, Forbes 1988, Semenchuk 1992), and unpublished sources
(e.g., colonial waterbird survey data for Idaho, Minnesota,
Wyoming). The cumulative total of 64,000 individuals does
not include non-breeders, which can be found along the 21
Pacific coast during the breeding season (Briggs et al~
1987, Campbell et al_. 1990, Henny et al .. 1990, Vermeer and
Morgan 1992). As.suming that the average recruitment rate for these grebes probably lies somewhere between 25 and 45%
{Johnsgard 1987),, a breeding population of 64,000, plus an estimated 5,000 to 10,000 non-breeders, would give rise to a total over-wintering population composed of no more than
102,800 individuals.
To evaluate the accuracy of this estimate, I determin(~.d the total number of Aechmophorus grebes reported from each of the five most recent National Audubon Christmas Bird
Counts ( 89th-93rd) • The large,st numbe.r reported was nearly
139,000, during the winter of 1989--1990 (LeBaron 1990).
Since the actual population size during the winter of 1989-
1990 presumably included more than 139,000 individuals, the breeding population estimate of 64,000 individuals apparently is low. Therefore, based on the Christmas Bird
Count data, I suggest that the current United States-Canada breeding population likely consists of somewhere between 120,000 and 200,000 individuals.
Individuals breeding in the United States and Canada winter mainly on the Pacific coast, from southern British
Columbia to extreme southern California (Root 1988, see below). Ratti (1981) counted Aechmophorus grebes at 16 sites in California during January 1977, and found that
13.3% of 2,049 grebes observed were clarkii. Using the 22 Christmas Bird Count d.ata from the five-year period mentioned above, I determined the relative frequency of clarkii reportc::'d from counts in British Columbia,.
Washington, Oregon, and California. Only those sites for which counts we:re· made during all five years, and for which the cumulative fiv·e;-year total of occidentali.s and clarkii was at least 100 individuals, were used.
A total of 75 sites met these criteria (see Fig. 3). I calculated the relative frequency of clarki.i for each 2° of latitude (Fig .. 3). For example, the sites between latitudes
34°N and 36°N had a cumulative count total of 16,150 individuals, with 7 .. 33% of the total being reported as clar.kii. The frequency of cl.a.x:kii declines with increasing latitude, from a high of 7.33% in southern California to a low of 0% near the Queen Charlotte Islands {Fig. 3).
Migratory Movements
Informati0:n O·n present-day migratory movements by
Aechmopborus grebes comes primarily from recoveries of dark phase individua.ls that were banded in Manitoba { see
Eichhcrst 1992). Grebes banded at two breeding colonies in Manitoba, a.nd subsequently recovered during winter, were primarily reco\tered along the Pacific coast (recovery-site locations extended from southern British Columbia to San
Diego, California). Although the relative level of breeding-area philopatry in the A.ec:hmopborus grebes is not known with certainty, information from the recoveries of 23
Figure 3. Relative frequency(%) of Clark's Grebes (Aechmophorus clarkii) reported from Audubon Christmas Bird Counts in British Columbia, Washington, Oregon, and California. Percent was calculated for each 2° of latitude. See Current Distribution and Abundance for explanation of how these% values were derived. 24
0 500 I I I
7.02% • 7.33;-\ •·. 38~N
~~~G··$ . 1r-~~---- 340~ 25 banded individuals, and multi-year observations of color marked individuals (Storer and Nuechterlein 1992; G. L.
Nuechterlein, pers. comm.), indicates that site fidelity is
not absolute, and therefore contemporary gene flow between
breeding areas potentially may be moderate to high. For
example, a grebe banded in June of 1977 at Delta Marsh,
Manitoba, was recovered at a site near Lac la Biche,
Alberta, in May of 1979 (Eichhorst 1992), a distance of
1,100 km between these two known breeding areas. CHAPTER III
MITOCHONDRIAL D'NA DIFFERENTIATION, POLYMORPHISM, AND
POPULATION STRUCTURE IN THE WESTERN-CLARK'S GREBE COMPLEX
(AECHMOPHORUS) OF THE UNITED STATES AND CANADA
Introduction
From 1886 until 1985, the Western Grebe (Aechmophorus occidentalis) and Clark's Grebe (A. clarkii) were regarded as variants or color phases of one species--the Western
Grebe (A. occidentalis; AOU 1886, 1983). Storer (1965) applied the terminology "dark-phase" and "light-phase" to them. The color phases are distinguished by differences in bill color and facial-plumage characters: (l) in dark-phase birds bill color is dull greenish-yellow and the black of the crown extends below the eyes, the lores, and the narrow line of bare skin extending from the eye to the gape; and
(2) in light-phase birds bill color is bright orange-yellow and the black of the crown does not reach the eyes or the lores (Storer 1965, Storer and Nuechterlein 1985). There is considerable overlap in all other plumage color characters
(Storer and Nuechterlein 1985) ~ In 1985, the light-phase form was restored to species rank (A. clarkii; AOU 1985) as originally described 127 years earlier by Go N. Lawrence (in
26 27
Baird 1858:895). The resurrection of the Clark's Grebe as a
"biological species" was prompted by observations of positive assortative mating between the color phases and the discovery of behavioral isolating mechanisms (see below).
Western and Clark 1 s grebes are widely sympatric in western North America from central Alberta south to the Mexican Plateau (Storer and Nuechterlein 1992, see Chapter
II). Two subspecies have been recognized within each of the
species: (l) A. occidentalis ephemeralis and A. clarkii
clarkii inhabit the Mexican Plateau; and (2) A. occidentalis
occidentalis and A. clarkii transitionalis occur within the
United States and Canada (Storer and Nuechterlein 1992).
The Mexican races are smaller in size than their northern counterparts (Storer and Nuechterlein 1992).
Western and Clark's grebes are perhaps best known for
their spectacular courtship displays. Of the 12 distinct displays performed by both species during pair formation
(Nuechterlein and Storer 1982), all but one is identical
between them. 'I'he single difference is the number of notes
in the "Advertising" call, one for A. clarkii and two for A.
occidentalis (Nuechterlein 1981a); but individuals of both
species have been found to possess the "wrong" Advertising
call (Nuechterlein 1981a, Eichhorst pers. observ.). This
behavioral difference, plus the above noted differences in bill color and facial pattern, allows individual grebes to recognize others of the same phenotype, and as a result 28 there is high (>95%) assortative mating (Storer 1965, Ratti
1979, Nuechterlein 1981a, Lindvall and Low 1982). Presumed hybrids (adults with intermediate bill-color and/or facial pattern phenotypes; Eichhorst and Parkin 1991} are found in many bieeding populations (Feerer 1977, Ratti 1979, De Smet
1987, Eichhorst and Parkin 1991) and are fertile
(Nuechterlein 1981a, Eichhorst pers. observ.).
Speciation models have been proposed by both Feerer
(1977) and Ratti (1977} to explain the evolution of the
Aechmophorus color phases. Their models are alike in that both invoke a vicariance event to explain the evolution of the two color-phase forms from an ar~estral Aechmophorus population. In Chapter IV, I review their models and evaluate their plausibility from a biogeographic perspective. I argue that Ratti's vicariance model is more unlikely than Feerer's vicariance model. In brief, Feerer's model suggests that the vicariance event occurred only about
6,000 years ago (see Chapter IV for a complete outline of his model). Additionally, Feerer (1977} hypothesized that intraspecific gene flow within the ancestral Aechmophorus population was high, a hypothesis which is plausible given:
(1) information on contemporary Aechmophorus migratory movements and breeding-area philopatry (see Chapter II); and
(2) the ephemeral nature of the lake/marsh habitats required by Aechmophorus grebes for nesting (e.g., Eichhorst and
Parkin 1991). 29
Studies of mitochondrial DNA (mtDNA) variation in birds have demonstrated the usefulness of using this maternally transmitted molecule for tracing phylogenetic relationships intraspecifically, and interspecifically where avian taxa are closely related (reviewed in Avise and Ball 1991). At the intraspecific level, restriction-enzyme assays of mtDNA variation have revealed a variety of levels of genetic population structure (Avise and Ball 1991); from species in which a single array of closely related mtDNA genotypes is geographically widespread (e.g., Red-winged Blackbird
[Agelaius phoeniceus], Ball et al. 1988; Downy Woodpecker
[Picoides pubescens], Ball and Avise 1992), to species in which multiple, genetically-divergent arrays of genotypes are associated with separate geographic regions within the range of the species (e.g., Seaside Sparrow [Ammodramus maritimus], Avise and Nelson 1989; Fox Sparrow [Passerella iliaca], Zink 1991). At the interspecific level, fixed rntDNA restriction-fragment/site differences have been found between several pairs of closely related species (e.g., Mack et al. 1986, Avise and Zink 1988, Zink et ale 1991b, Gill et al. 1993.). One notable example is the assay by Avise and
Zink (1988), of mtDNA divergence between the Long-billed
Dowitcher (Limnodromus scolopaceus) and the Short-billed
Dowitcher (L. griseus). They found that these "sibling species" had differed by at least 24 restriction-site differences and an estimated mtDNA divergence level of 8.2%. 30
In this chapter, I present the results of an analysis of mtDNA variation in the Aechmophorus populations of the
United States and Canada (i~e., occidentalis and
transitionalis). Using restriction-enzyme analysis, I assessed the level of: (1) mtDNA divergence between the two color phases; (2) rntDNA diversity within each of the color phases; and (3) mtDNA diversity within and between
Aechmophorus breeding populations. Because individuals of the two color phases do hybridize, a sample of intermediate
phenotype individuals (i.e., presumed hybrids) was included
in the survey. I predicted that if Feerer's vicariance model is correct, and gene flow was high between breeding
areas within the historically-recent ancestral Aechmophorus
population, then: (1) there would be little or no
differentiation between the mtDNA genomes of the two color
phases; and (2) by extension, there would be little or no
geographic structuring of observed mtDNA genotypes among
Aechmophorus breeding areas within the United States-Canada range.
Materials and Methods
Choice of sample populations and field collections.--A
sampling design in which grebes were collected from
lakes/marshes within a southwest-to-northeast geographic
corridor, representing three arbitrary geographic regions of
the United States-Canada breeding range (southwest (SW],
central [CN], northeast [NE]; Fig. 4), was used since a high 31 proportion of Aechmophorus grebes apparently nest within the corridor (see Chapter II). During May-August, 1989-1991, I collected grebes at seven breeding sites within the corridor
(Fig. 4). Collecting in the central region of the corridor was restricted to one site (MANR) in Utah, since it was the only state in that region for which I was able to obtain a collecting permit; I found MANR to be the only site within
Utah with an Aechmophorus population large enough to provide
a sufficient number of specimens.
Both adult and young grebes were collected. Most grebes were collected by shooting, but some {mainly small chicks) were captured alive and then sacrificed. Adults
were identified as A. occidentalis (WN), A. clarkii {CL) or
intermediate between occidentalis and clarkii (IM; i.e.,
presumed hybrid) based on bill-color and facial-plumage phenotypes (Sto=er and Nuechterlein 1985, Eichhorst and
Parkin 1991). Young grebes were classified based on the phenotypes of the adults attending them. Hereafter, I refer
to WN, CL, and IM collectively as Aechmophorus phenotypes.
I was unable to obtain any known hybrid chickso Tissue was
obtained from a total of 241 adults and 32 chicks/juvenileso
For most specimens, only liver tissue was removed, but for
small chicks, liver, heart, and kidney tissue was saved.
All tissue samples were stored in an MSB-ca++ buffer
(Lansman et al. 1981) and frozen in liquid nitrogen until they were transported to the University of North Dakota 32
Figure 4. Aechmophorus breeding sites within the
United States-Canada breeding range {stippled area), from which specimens were collected for mtDNA survey. Breeding sites: Alkaline Lake (ALKL}, Kidder Co. [R70W, Tl37N], North
Dakota; Delta Marsh (DELM), Manitoba; Eagle Lake (EAGL),
Lassen Co., California; Goose Lake (GOOL), Modoc Co.,
California; Lake Osakis (LOSA), Todd Co., Minnesota; Mantua
Reservoir (MANR), Box Elder Co, Utah; Upper Klamath Lake
(UKKL), Klamath Co, Oregon. Geographic regions: southwest
(SW}; central (CN); northeast (NE). 33 ,i~ '\·, '""·~"'·,~~ \ -~ ... -......
~ ~ ~
500
' KIiometersi.-' 34 where they were stored at -90'°C. All specimens were shipped
to the Zoology Museum, Uni.vers.ity of Michigan to be prepared as flat skins and.skeletons (adults) or study skins (chicks) for additional studies of plumag·e and morpbometric variation
( B.ichhorst and. R "'W &· Storer in prep. ) ..
La.bo.ratory analysis.--Mitocbondrial DNA wa.s isolated from tissue samples using a. s-u.c-rose step-gradient protocol
( Spolsky and Ozell 1984) a.s modified by Tar.r ( 1991} ... Mitochondrial DNA samples were digested to completion, with restriction enzymes, under temperature and bu.ffer conditions specified for ~ach individual. enzyme by 'the manu.facturer.
Resulting m.tDNA fragments were end-labeled with appropriat.e
;.i;iP-tagged nucleotides using 'the la.rge {Klenow) fragment of
DMA. polymerase I, and separated by electrophoresis through l. 0-1 .. 21 aga,rose gels.. A -molecular size standard ( 1-~ ki lobase [. kb J ladde.r purchased from Bethesda Resear·ch Laboratories) also wa.s end-labeled and run on each gel to determin.tl the sizes of the mtDNA f·ragments. Gels were dried on 3MM Whatman paper under vacuum and fragments were
rev"ealed by autoradiogr.-aphy ..
Gels were run so ·that all fragments greater than O •. 3 kb i.n si~e would be detec'ted.. When the existence of fragments 0 .. 2-0 .. 3 kb was suspected (based on the additive loss or·gain of appropriately sized. frAigm.en.ts), additional gels were run
for a shorter period of ·time to conf inn their existence. I was unable to detect fragments less than 0.2 kb in size with 35 the agarose gels, but few fragments of that size were suspected to exist. The mtDNA fragment patterns of the autoradiographs (including the 1-kb ladder) were scanned with a Hewlett Packard Scanjet Plus, imported into the Collage~ 2.0 (Photodyne Inc.) software program for the Macintosh, and the size of the fra.gments was estimated with the program's calibrate features.
In preliminary surveys, a total of 37 restriction enzymes was screened using a minimum of 10 individual mtDNA samples (both WN and CL represented) for each enzyme. Those enzymes that produced patterns too difficult to reliably interpret (HhaI,. MboI, MspI, NciI,, StyI), that produced fragments whic.h did not end-label consistently (ApaI, Bg1I,
EaoT22I, PvuI, SacI, SacII), or that produced less than two fragments ( A.f1II, BanIII, BspEI, Bst:EII, KpnI, MluI, Pst:I, SalI, XbaI, XhoI) were not used for further analysis. The remaining· 16 enzymes ( ApaLI, AseI, Ava.I, Ba.mHI, Ban!, BclI,
BglII, BspHI, BstBI, BstYI, BcoRI, HindIII, NcoI, NdeI,
NheI, SpeI} were u.sed in the final survey. Preliminary surveys indicated a low level of geographic variation, so mtDNA was isolated from only 140 of 277 available Aecilmophorus grebes to save time and reduce expense. Of those 140 mtDNA samples, 109 were sufficientl:y- "clean,•• and comprised the final sample for the study.
Data analysis.--For each enzyme, the most common fragment pattern was assigned the letter C, the next most 36 common pattern the letter A and the least common pattern the letter B (no enzyme produced more than three patterns). The mtDN.P1. genotype ( haplotype) of each individual grebe was then a composite of the letter codes for the 16 enzymes ultimately used (see above) in the survey. Using estimates of fragment sizes, restriction sites were inferred from the fragment patterns, and scored as present or absent for each individual. The simple structure of the mutational transformations between patterns (see Fig. Sa) precluded the need for formal mapping of sites, thereby allowing all data treatments to be based on mtDNA "site" rather than
"fragment" data.
Numbers of nucleotide substitutions per site (d) between haplotypes, nucleotide diversity (1r; Nei 1987), and nucleotide divergence ( dx}· and dA; Nei and Li 1979) were estimated from the inferred site data. The RESTSITE 1.2 software package (Miller 1991) was used to calculate these values along with their standard errors. Details of the methods and equations used in RESTSITE are given in Nei and
Miller { 1990) • '!'he matrix of d-values between haplotypes was used to phenetically cluster the haplotypes using the unweighted pair-group method with arithmetic means (UPGMA;
Sneath and Sokal 1973). The UPGMA option in the RESTSITE package was used to conduct the cluster analysis. Dolle and
Wagner-parsimony analyses of the restriction-site data were done using the PAUP 3.1.1 software package (Swofford 1993). 37
Figure 5. Unrooted parsimony network sununarizing restriction-site changes between the 12 Aeahmophorus mtDNA haplotypes. (a) Fragment patterns [A-C] produced by the enzymes listed. Direction of arrow indicates inferred restriction-site loss between adjacent fragment patterns.
(b) Most parsimonious, unrooted network produced by both
Dollo and Wagner parsimony methods. Inferred site changes indicated between adjacent haplotypes. 38 (a) Asel C - ... A HindIII C .,. ·A Aval C .. A BamHI A .. C ... B BspHI A .. C .. B BstYI A .. C .. B (b) Nhel C .. -B ... A Spel C .. A .. B
Nhel B
NheI A
Asel I BspHI 39
The branch-and-bound search option was employed, using the presence/absence of sites as characters.
Haplotype frequencies were compared among phenotypes, among breeding sites, and among geographic regions using Chi-square tests. Because some cell frequencies were less than five, significance of calculated Chi-square values was
determined using a Monte-Carlo simulation (Roff and Bentzen
1989) with 1,000 random shufflings of the data (REAP 4.0
software package; McElroy et al. 1992). Haplotypic
diversity (h; Nei and Tajima 1981), was calculated as
h = ( n In - 1) { 1 - tx/) ,
where x 1 is the frequency of the ith haplotype in a sample of n.
I assessed the degree of within- and between-population
genetic variation using two methods. Since there was no
detectable mtDNA difference between A. occiden~alis and A.
clarkii (see Results below), and the distribution of haplotypes was homogenous among geographic regions within
occidentalis (X2 = 21.19, P = 0.246 ± 0.014) and within
clarkii (X2 = 10.04, P = 0.152 ± 0.011; only SW and CN were
compared due to small sample for NE), the phenotypes were
pooled for the analyses of population genetic variation.
The first method involved the estimation of interdeme
genetic variation (G5T; Nei 1973) using the approach of Takahata and Palumbi (1985). Unlike Nei's method of
estimating GsT, which simply treats haplotypes as alleles, 40
Takahata and Palumbi's method treats each restriction site as a separate locus with two alleles (site is present or absent), thereby allowing distinction between closely related and more divergent haplotypes. This is accomplished by estimating intrademe (I) and interderne (J) identity probabilities from a presence/absence matrix of restriction sites in each deme. Essentially, I and J are the probabilities that two homologous mtDNA molecules randomly sampled from within and among two demes are identical by descent. These independent indices of intra- and interdeme identity are then used to estimate the interderne component of total genetic variation (GsT). Identity probabilities were calculated according to equations 17 and 19 of Takahata and Palumbi ( 1985) . 'rhe means of I and J were used to
calculate average gene diversity of dernes (H0 ) as
and average gene diversity of the total population (HT} as
HT = 1 - [ ( 1/Limean) + ( 1 - 1/LJmean)], where Lis the number of demes. The amount of mtDNA
restriction-site variation due to interdeme differences
( Gs•r) was defined as
GsT = ( HT - H0 ) / HT.
Since the variance of the ~T statistic is not known, the
approximate significance of observed G5 T values was evaluated using the bootstrap method as adapted for GsT by
Palumbi and Wilson (1990). A computer program provided by 41
S. Palumbi was used to perform the above calculations and bootstrapping. I estimated GsT for the group of breeding sites and for the group of geographic regions.
The second method involved the analysis of molecular variance (AMOVA) approach of Excoffier et al. (1992). Their method incorporates information on haplotype divergence
(interhaplotypic squared-distance matrices) into an analysis of variance format, producing estimates of variance components and F-statistic analogs (termed I-statistics).
Several alternative matrices corresponding to different types of molecular data, as well as different types of evolutionary assumptions, can be used to produce variance component and t-statistic estimates for different levels of hierarchial subdivision. I used two matrix types in the AMOVA analysis. One matrix consisted of the number of restriction-site differences between the 12 haplotypes
(i.e., phenetic distance metric), and the second matrix was made up of weighted evolutionary distances (dxr), measured along the haplotype parsimony network (Fig. Sb; see
Excoffier et al. 1992 for a discussion on matrix types and their use). For the Aecllmophorus data set, the AMOVA hierarchial model consisted of 11 within breeding sites,"
--among breeding sites/within geographic regions, 11 and "among geographic regions" components of diversity. The CN region was not included in the analysis because only one breeding site had been sampled in that region. Significance of 42 variance components and ~-statistics was assessed by generating null distributions from 1,000 permutations of the input matrices {Excoffier et al .. 1992). A computer program provided by L. Excoffier was used to perform the AMOVA analyses and generate the null distributions.
The evolutionary effective population size of
Aechmophorus females ( Nfre,) was estimated using the simulation approach of Avise et al. (1988), with the assumptions that mtDNA evolves at a rate of 2% per million years in birds (Helm-Bychowski 1984, Shields and Wilson
1987a) and that the average generation time for Aechmophorus grebes is five years. Although generation time is accurately known for few species of birds, for those species in which it has been determined, the observed high correlation between the age at first breeding and generation time suggests a ratio of 2.0 to 3.0 (Sibley and Ahlquist
1990). Aechmophorus grebes probably can breed at one year of age (Storer and Nuechterlein 1992), but based on life span estimates of banded individuals (see Eichhorst 1992), most individuals probably breed for the first time at two years of age. Thus, based on these ratios, I consider five years to be a reasonable estimate of generation time.
Results
Mitochondrial-DNA genome.--The Aechmophorus mtDNA genome varied in size from approximately 18.7 to 19.4 kb.
In addition, of the 109 individuals I surveyed, 102 were 4.3 detectably heteroplasmic for size variants (i.e., possessed
two or .more genomes that varied in siz,e) ; one individual
a.pp~at'~d to pos9°~ss. 10 different ~12:e genoffieg. .. Siz~
estima·tes of f ragment..s indicated that the obsexved intra
and interindividual size polymorphism could be explained by
the presence of a tandem repeat approximately 0.06 ta 0.08
kb in size.. A O. 07 3 kb tandem repeat rece,ntly has been located in the control region c--o loopn) of the Aechmophorus mtDNA genome (T .. w. Quinn, pers. comm .. ).. 'l'he presence, in some patterns, of more than one series of homologous size variable fragments, however, suggests that this tandem
repeat may not be confined solely to the control region.
Quantitative assessment of the frequency of observed
size variants was not feasible because of the high level of
heteroplasmy and because of variability in fragment resolution. But, a cursory assessment suggested the
possibility of homogeneity in the relative frequency of size
variants among the Aechmophorus phenotypes, among the
breeding sites, and among the geographic regiDns.
I also detected intra- and interindividual mtDNA size
polymorphism in three other grebe species (eared [Podiceps
nigricollis}, red-necked [P., grisegena], pied-billed
[Podilymbus podiceps]). To date, mtDNA size polymorphism
and heteroplasmy has been observed in a limited number of
avian species (Avise and Zink 1988, Ball and Avise 1992) II' 44.
'f'he 16 enzytfilfJ:s: 1 employed produced a total of 89 detectable rest:,rlction fragme·nts from the 109 grebes that I
surveyed jl In ,. 76 :res.tric,tion site·s were inferred from the 89 fragments (see Appendix), and on the average 69 sites wet·r:.- scored for each g·rebe, ( :repres,enting about 2: .1% of the
ze-,rariabl,? mtD'tlA genome)" Each of e.i.ght enzymes (ApaLI,.
Ban!, Bell, Bgl'II I BstBI, BcO:RI, NcaI, NdeI) produce.cl only a single fragment pattern. 'Jlhe other eight enzymes each produced from two to three patterns (Table 3), revealing a total of 13 restriction s.ites wh.ich were poly:morphic (i.e.-, present in some individuals and absent in others), with all mutational transformations easily explai.nable by the gain or loss of single sites (Fig. Sa.). R.estr·i.ction·-site heteroplasmy was not observed in any of the: individuals surveyed .. Frequencies of the commonly observed fragment patterns varied from 0.98 (Asel) to 0.79 (SpeI) with none of the patterns being fixed for ocaidentalis or clarkii (Table
3) •
Haplotype phylogeny and diversity.--Twelve mtDNA haplotypes were detected among the 109 grebes surveyed
( Tables 4 and 5) . F'our of the haplotype.s where restricted to single individuals .. The most common haplotype (il) was shared by 49 .. 5% of the individuals .. Haplotype frequencies were hornogenous among the Aechmophorus phenotypes (X2 =
18 ... 83:, P = 0.638 ± 0.015) .. Differences between any two haplotypes could be explained by one to four site m.tDfJA ·f :ragm*.:mt pat:.terns; -restr,,i,.:rt:ion si:tes: ,,er·e of 109 A.echJtt,OphO/JtUfS,'
Rest:rict Fragment WM CL IM .All 1r enzyme pat:ter.n { .n•«H) ( 11*38) (nlf:lf::1,0) Ii t:rm'~~~~.-ltit~~~64,f.U~..f;:t;.'2;(£_..~~'*1Jilill.l~~~.wif~~Jl~lillU~$1lib~JI? .. wa•~~"""""'liiior~~fl?'i'· Asel C 1 •. 00 0~95 lit:00 o •. 9s A 0 0.,05 OJ o ..
Aval C 0.,90! 0.92 1.00 o.. A 0*10 0.08 Ol Oll,
BamflI C 0~96 0.9.5 0.90 0.95 A 0.02 Olti05 0.,10 0 •. 04 B 0;,;02 0 0 0.01
Bs·pHI. C 0.98 0.92 14i100 011c96 A 0 Ot08 0 0".03 B 0.02 0 0 0 .. 01
BstYI C 0.,88 0.,87 O RindIII C 0.95 Q.95 141.00 0.95 A 0,.05 o.os 0 0.05 NheI C 0.85 0.87 0"'80 0.85 A 0.08 0.,13 0.20 0.11 B 0~07 0 0 0..,04 SpeI C 0.,,83 0.71 0.80 0 .. 19 A 0.15 0.29 0.20 0.20 B 0 •. 02 0 0 0 .. 01 ~ WN .:::; A... accidentalis; CL :=.: A. c:larkii; IM ::; intermediates .. .All 1l ff» !Si 2 ~ i·n :ai. J 5i ,] 2 :Hll 4 l ,!. !;. 5 .l 1 4 6 l 2 l 4 '1; 4 2 2 9 '] l ]l l 1 1l Jt 12 :u .l 61 109 • Wtl 5 ll@; ~ A~· c la:ckJ,,:1. t U'C •· in t,erm~dia t;Jim: ., ~. xtigbt, refe:X· ·to .f:ra~mtt. pa;t't,erns enz;y.ffflies: AS?el, .Aval ,. BaJmHl ,. I~ peit ~d te ( d) be·t'fwttn all pairs of mtJlNA 6 ~· Pairwise distances ranged by t.he UP!3tm. 1c1·avealed no ffl!ajo.r.· cl·ii:tsters of hc;iplo:typ~s; (Fig., G).. P,ar·s:imony analyaeft rervea1-led lit::t.le or Table 5,., Geographic of necnmopnoru ~111,;h==,-· i!!l-c. -- ';"--ec~z-«.- ·--- ~ ---··-· --- ~ ~ - --··· -~ ~. ~ £f:!f5'1?:~ ,B reed .ing~ si~*" ~es .• Haplotyp~t' ALKL DEbf.! LOSA E.AGL NE ~·'cl 1 cccccccc 11 8 4 8 8 9; 23 8 4J 2 CCCCCCCA l l 2 5 5 4 4' 4 s 3 CCCCCCAC l 1 J 2 3 2 3 s 4 CACCAACC l J 1 l 4 ~ 5 C.ACCACCC 1 1 1 l 1 1 4 l 6 CCACDCCC l l 2 l * 2 "} 7 CCCCCCBC 2 2 2 £, ~ B ACCACCAC l l l 1 ~ 9 CCBCCCCC l l 10 CCCACCC= l l 11 CCCBCCCC l l 12 ccccccca 1 l Total 16 11 8 20 1""'I 18 19 35 20 54 ~ L~tt~r codes r~fer to breeding Git~~ ~nd q~ographie r~gi.on~ &hown ltl Figurft 4~ h Geographic rftgions include bret1ding 3it~s as follow~; lffl (AL~L" DJUM, L-OSA); CN (MANR) J SW (UKLL, GOOL, BAGL). · ~ Letters, from left to right, r@for to fr~gment pattorn6 {A-C) produc~d by restriction enzymeo: Asel, Aval., BamHI, BspBI, BstYI, B1ndIII t Nh.~I 1 ~peI ~ Table 6.. J=;p;timate1} of n~T.ber of nucleotide substi tut'.ions p~r site :mWN..,:t\ h~plotypes ob~erved in l 09 Aechmopboru.s grebes. d-values are above the errors belO\aJ the diagonal. All v~lue.s e:xpre~Ged as perc@nt. see Table 4 for q,enotvng;s l 2 3 & 5 6 7 8 9 10 11 12 1 0.1262 0.2481 0.3788 0~2564 0.2553 0.1243 .S014 0~1243 0.1262 0~1262 0.2518 2 0.1212 ==== 0~3769 0.5082 0.3845 0.3866 0.2518 0~6350 0.2518 0.2557 0-2557 0.1262 3 0.2511 0.2762 ---- 0.6221 0.5007 0~5057 0~1225 -2444 O.llll 0.3769 0.3769 0.5014 , 0.1933 0.2043 0.2935 ---- 0.1243 o~tt7o o~soo1 0.11&2 o~soo1 o~soaz o.soaa o~&ll4 5 0.1729 0~1949 0.2872 0~1254 ---- 0.5235 0~3788 0.7501 0.3788 0.3845 0.384S 0.5082 6 0.1102 0.2115 0.3076 0.3125 0.3093 0.3808 O~l6S5 0~3108 0.3866 0.3866 O.SllS 7 0.1258 0.1739 0.1238 0.2158 0,2023 0.2110 0~3713 0.2411 0.2511 0.2518 O.l769 ~. 8 0.2819 0.2876 0.1595 0.2743 0.2834 0.3140 0~1878 0.6254 0.3769 0.6350 0.7603 00 9 o.12s9 o.11ss o.2aoa 0~2193 0.204s 0.2134 0.1114 o.l014 ---- o.2s11 o.2s1a o.37&, 10 0.1212 (Ll703 0.2762 0 .. 2043 !LUJO 0 .. 2115 O.l1J~ 0.210, ~L17SS ---- o.:as~1 0.3827 11 0.1272 0.1703 0.2762 0.2043 0~1949 0~2115 0.1739 0.3514 0.1155 0.2581 ---- 0,3827 12 0,2539 0.1272 0.3452 0.2765 0.2767 0.29Sl 0.2756 0.3409 0~2775 0.2722 0.2722 F;tgure 6. UPGMA cluster analysis of the 12 Aecbmop,ha:r:us mtDHA haplotypes: ~ I l I 9 ... i 10 :ll -: I 3 I : 1 I. 12 I 2 6 I 4 I s 8 I_ 1 I _ WV I I - f I I I 0.7 0.6 0~5 0.4 0~3 0.2 0.1 0.0 Percent Se,quence Divergen,ce 51 Haplotypic , which take into account the nmobe:r haplo,types within samples: as we:11 as thei.r relative frequenc· , are given in Ta.ble· 1. Hap1crtypic diverf;:ity gives the probabi.lity that two randomly drawn .individual.a: from a sampl.e b.ave di.f:fe:rent :mtDIUt genotypes, and ls e-quivale.nt to, heterozygosity or gene diversity ur~ed in studles: of p:rote,irJ polymorphi.s.m (Mei 197 5) .. Haplcrtypic diversity waa: 0 * 142 in clarkii and O .. 680 in occ.identa,lis. 1 values which are comparable to estimates .for other avia.n species: (Table 8) ~ Haplotypic dive·rs.ity varied f:rom O" 561 tc, 0. 784 trt the g:e.ographic-.reg'i.on lev:3:l and from 0.473 to 0.819 at the breeding-site le.ve'.l.. For the NE region, the difference bet.ween 'the breeding sites with the highest (LOSA) and the lowest diversity {DELM) was 0 .. 277, more than twice the comparable di.fference for the SW region (0 .. 121} .. Nucleotide d1.ve·rsity and divergence. --Nucleotide diversity ( ;i,r) is an estimate o,f the p.roportion of nucleotides that differ be:twee,n two genotypes randvmly drawn from a sample. Estimates of Tt for the, Aechmophorus phenotypes, breeding sites, and geographic regions are given in t'.I'able 1., Nucleotide diversity for the entire sample ( O .18%} falls within the range of e,stimates fo,r other avian species (Table 8). When there is DN.A polymorphism within a population/species1; the .;:::.xtent of net nucleotide divergence r!fable 7 ·V f~;13:tiJrm11te1; mt.DMA m:icle:ot:.ide: ( 1r.) and haplotypic , h} diversity Aechmopborus grebe·s s;urve:ye.d in this study" 1r exJ,,ressed as p,Enr·c:ent •. n n i (SE) h i; ( SJ~) ,':,;:"~.,,..=-=·--.c.;· ~~,1./'. - wr. 11 Phen,o.t.ypes · WN 61 0 .. 1634, (.0".0618} 0.680 ( (). 059) C.L 3:8 0 •. 2190 (0 .. 0692.) 0.1'42 (0.051) IM 10 0 .. 1838 (0,.1006) 0.1133 (0 .. 120} ·sre;eding· siteab ALKL 16 0!11517 (0 .. 0582) 0 • .54,2: ( 0. 14 7) DELM 11 0 .. 0635 (0 .. 0457) 0.473 (0 .. 162) LOSA 8 0.1196 (0.0873) 0 ... 750 (0.139) MANR 20 0. 2141 <0 •. 0795) 0 .. 784. (0.067) U.KLL 17 0 .. 1904 (0 •. 0771) 0 .. 698 (0 .. 075) GOOL 18 0 •. 2005 (0.081'9) 0 .. 765 (0.081) El\GL 19 0~2221 (0.1006) o ... 819 (0 .. 063) Geographic regions:h NE 35 0 .. 1277 {0.0549) 0 •. 561 (0 ... 096) CN 20 0.,21.47 (0.0795) (L, 784: (0.067) SW 54 0.2112 (0 .. 0725) 0.755 ( 0. 04,5) ALL 109 0~1840 (0.0659) o.:ios NA • WN = A... occiden'tali.s; CL = A. clarkii; IM = intermediates. h Letter codes refe:r' to breeding sites and geographic reqions shown in Figure 4: .. (d11 ) between any two closely related populations/species is measured as where d,'f ~md dr are the 'Jr: values for population/ species X a.nd :t, and d.>as is: the average murJ)er of nucleotide substitutions per site between X and Y (Mei and Li 1979). Intraspecific poly1norphis.m is thus subtracted fr,om the total Table 8. Estimates of mtDNA polymorhism within North Am.erican avian species. Some valuea not provided in original sources are from Table 3 of Avise and Ball ( 1991}. Number of mtD:NA restriction Nucleo- Haplo- lbird:s haplo- sites or tide typic :sur- types fragments di\:fer- diver- s"pec1es I vey1ed fou:nd per bird sity sity Sourc~~ Jlechmo_phoru.s clarkii. 38 :a 69 0~00122 0 .. 142 l A" occtdsntalis 61 10 69 O.OOil6 0-.60·8 l Branta canadensis 53 11 183 011'0·07 Q: .. 861 2 Chen caerule~cens 129 .11 81 0~006 0.694 3 ~ C~ ros1JJi 31 2 81 0~002 0~181 3 w Anas platyrhynohos 20 7 93 0~004 O~S.05 4 A. rubripes 20 3 93 O·. 001 0*489 4 Agelaius pho.t~niceus 127 34 63 0.002 0.833 s Ammodramus maritimus 40 11 89 0.,006 o... 7'09 6 Parus major 18 13 204- 0"0019 0 .. 980 7 Carduells flammea 31 17 109 0.0021 0~802 8 Passe,r: iliaca 47 5 140 o.oos 0.720 9 M~lospiza melodia 27 15 96 0 .. 002 0 .. 909 9 Quiscalus q·uiscalus 35 29 96 NA 0.973 10 PicoidtuJ pubescens 51 5 63 0 !t O(l03 0~152 11 Molothrus ater 26 6 NA 0.0011 0 .. 671 11 Zenaida maaroura 17 4 NA 0.0011 0"331 11 Pipilo erythrophthalmus 19 5 NA 0.0034 0.708 11 Geothlypis tricbas 21 10 NA 0 .. 0048 0 .. 776 11 Table 8. Continued 4 {1} this study; (2) Van ¥,agner and Baker 1990; {3) Avise et al.. 1992; {4) Avis~ et al~ 1990; (5) Ball et al .. 1988; (6) Avise and Nelson 1989; {7 Te·gelstrom 1987; {6 Seu-tin 1990; ( 9) Zink 1991; ( 10) Zink et al. 1991c; ( 11} Ball Avise 19·9·2 .. ~ ~ f l eiot.ide, tnllbs:titutions per s:ite'. ( '111 i ~ tlet should be d.et.ffr.mined in those ca~e,si where t5;peci0'M are :rect?n.tly a;epa1:·a.t:.,,,J, sd.. nce i.t. ls likely that rnucn of the 01b~:e:rve;d mtDN:A variation .i.s due to po,lymo:r1,hi~.ms that aro~e in the ant.!'e.s:t:r·al po)pJtd.at.ion, hut which are &till present in the daughter species~ E~ti.mate~ o:f and d.41 be·tween the .Ae:';'lhmopborus phenotype,a:, between the br·e.edlng a;ite~, a.nd bet:ween the geographic regions are given i.n Tables 9, 10 ,- and ll"' Half of all tht? of net di·lte:rgenc1e { d,i\\} consisted of values that were negative .L, id.gn (T1ablen 9 and 11).. If two populations/ a:n1:. because the standard errors calculated 'for· the iriividual d4 estimatr~s ( not s,hown) were, larger than the corresponding dA value in half of the ca.ses.. The total number of restriction site.J, generated by my assay appa:r·ently was not sufficient for accura.tely detecting levels of net, di'ver9e.nce that truly may have been close to zero. Popu:latJon g·e.netic va:riation.--Geographic distribution of the Aecbmophorus haplotypes (Table 5) was homogenous among the breeding sites(~= 65~08, P = 0.560 ± 0~016) and a:mong the geographic :regions ( X2 = 19. 071, P = 0. 10.3 :! 11!1llft 1i)!~ iN, n·1i:1JJ111;b1£tr Bet.,.•o:n. .Pb•not.ype11t11 W?J vcr~1-1.~ 0 ~, 191,) ·t O .,, 064.S -·O ,,, 0003 ·~m veri,us; U1 Oi ~ l69J9 .'! 0. @1'f)I] ..,.,(()M\0031 CL vet~stu, IM oi.~1954 i (J1 .. (0i'J$1 -·G~ Ol06,4 Jlotweein g·eagr&.\l'ld.c regi,on•~· 0 tfE vc1 r: s; us, CH O~lG90 t 0..,0646 - 0 ~ 0022 HE versus: SW 011, 1708 t O~, Of,;;}'7 0110014. versua 0 206S t; 0. 0131 -01,.,0064 ll'l WH 'IW, A. intermed.iates .. tJ Lett.er F'igure 4. geographic for the breeding sites {Table 12)., In:te:rdeme ideJltity probabilitie~ (-.7) lik.e:~liise: "~ere high {HE versus sw~ o. 900; CN versus. swt 0 .. 9091 NE: versus, cu, 0.911; see Table 12 for· breeding sites}. The amount of m:tDNA sit.e var·ia.tion due t.o inte·rde:me: dif:ferences ( Gg,) wa.s liini.te.d between the t.hree gE:-1ographi.c regions, { 12%) an.d betweem t.he seven breeding sites ( 23%) -· S:i.gni·ficant ~,nterdeme :,e:netic structuring wa.s not indL::::ated for either category, as obse:rved G$1f values we.re: simila.:r to the randomly-generat.ed G:s,~· value,s ( not shown)~ 'I'able 10. Average sub :s,tr1C.U. • 11,,c t'_ions p~r si~t -e (, above diagonal; SE d.iagoual) Afl'ci1RKJpborus breeding 4 ~ites .. All va.lues eltpr«~rs:sed. as perco:Jlt"' !Kt! l!l!L _F,.R'!\iL,_ . .,~h. L~!l OELM LOSA OKLL BAGL =-- - ~ P.,.LKL ~~~:,,,,., (L 9 0 .. 1604 ~l 0 .. 1841 0~,116S 0~ 6 DELM tL ~"""'~ ..... ~ 0~1167 0~ O~ 53 0~1341 0. 146,4 LOSA 0~064.0 0~ (L, 1852 O~ 1941. 0.17'4() 0 .. 19G2 MAtU\ 0~063S 0~0607 0"0741 0~2050 0.,1995 0~2145 UKLL 0~0515 0~0525 o.~0667 0~0670 .,.,319~f,-it:~ 0$2030 0~1257' \,ft GOOL 0-0663 0~0638 0~0793 0.0782 O,t0118 ~:sm,:i:,,,t,411!1 (\~212Ji ~ EAGL 0.0790 01'0754 0.08·60 Oot086l 0.0756 0$0811 .,.. .,,.~.,.,.. ;J Lettf}r cod~~ r0fer to br@~ding 3i't~~ ~hown in Fiqur~ 4£ ~rable Jl lL.,, .Ave:rraicg1e l1lJtll!C le,o,t;. ide s,u:» bsrt.. iL t. taJt:iL o)rn s, ~it~ «d1tJ bg,t.~P®i~:Jl'll ~;i·t.e:ia;lf!.. .All ,,.a.lu.e,is ex:p:r·t:u1t11111d a§ p~r·c;"1r,·t.,, ,., (H)2'1 -0,;,0052 1 "' tOLll 54 ,.,(0) .. @C)) 8 Jl (OJ ~ Qi O16l E.AGl.A -·O ,., «MH6 -,@ •. O«JJl9 (0) ,,, ~194 neii1:t:i:1Ly iden·t:icaJL :rea'tt.lll.'t:si; mtDltA roa,t.ricrtJLoi:ftl-·adUte ·vaxiatlo)n wa9 found 'f'htt ob!ie.rv·ed ~ax'iance comrp;0:nen'ts f 'r·o,'Cl11J v1>l't!i«?tt, gentn:ated by :m!lltr'.'ilce.6 ( llfaiJble 13) ., atatu:• o:l Ute colo,r pll••••~-··"'·'""1 ~eige:l and Avl~ti fl 6) tbe e:vol urtloiruury dy~a.mic:s (Ji'ff fel'ffl1tde· l @Ul" 0 1£ daught.(n;:· 1rpe:c.i.e1s ;from an. 11'he\y found t.hat. :f o,l.lowin,g { s1uch a:s; by a geo;graphic or 59 ~, 12.,, I.nt:1ra1d«:!\C1me idernitity probab.ili:t.ies ( 1111ndl.e:rlined) for Aecbm .A1L,K:t LO.SA M.1\ttR 0 .. 931 Q .. ll:l 0,,, 0,.,935 _!))~_64 0 .. 9:18 0.920 0 .. 92~~ BAGL 0 921 0.,923 0 .. 924: 0 ... 908 ) , pbyloge:net'..iC: s:ta"t.us of a given pair t.o nttt:J)1NA) progresses ·through a common 1 in d.aug:hter po.piula.tJ,.on ""S/" 'tha.n they a.re t.o other in 1 o,ver time a:nid new m.utaticms lineages, one of the daughter all Table 13~ Hierarchial analysis of molecular variance (AMOVA) on two matrices of mtDUA g-enetie distances between Aechmophorus haplotypes ( see Materials a.nd Methods for explanation}.. Only Sid (bre·eding sites EAGL, GOOL, UKLL} and NE (A:LKL, DELM, LOSA) <}eograpbic regions used in analysis .. % of ~ Variance component Variance, total r statistic~ ~ . Based on si'ta differences i\.lfiong geog rap hie reg ionfi 0 .. 0126 1.74 00)35 lt·~i· e O4 0 l 7 Among bre,eding Gite~/geographic regions 011-0046 0,,63 0.21 t~~ $, 0 .. 006 Within breedi~g aites- - - · 0.7090 9.,L63 0~26 t,~'r • 0. 024 Baa:ed on pDre-imony .network Among geo,9xaphic regions 0.0132 L82 0.39 t,'f Bk O't 018 Among bre,eding tiites/geographie regions 0~0033 0.45 0!!30 '~~ ~ o.. oos ~ Within breeding aites 0~7102 97.73 0!,23 ts~ & 0 • 023 0 ~ Probability of getting Q r4ndom $tatistic (i.e. varianeet t) strictly larg@r than the observed statistic; based on 1,000 random permutations of the data set. Probability random breeding site heteroscedasticity > observed heteroecedasticity §1 0~287 for both site-differ~nce and parsimony-network variance comparisons. Probabilit}' random geographic region heteroscedaaticity > observed heteroscedasticity s 0"056 a;nd Q,,064 for site-difference and parsimony-network variance compariaons, respectiv~ly~ 61 ancestral lineages except one (L,e·..,, the: haplotypes within 1'A'' thus make-up a mo:nophyletic group,}., Population na"· will thus be paraphyletic with respect to ~A.p Eventually, population '11 B" will al.go become 1nonophylet.ic, and the daughter populations thE,-m have become reciproca.lly monophy1 etic in matriarchal anc.est:cy .. The phylogenetic status of a givren pai.r of spe,cie.s is defined by the relationship between maximum gem?·tic distances among individuals with.in each of the two s.pecies,. and minimum genetic distances between individuals of the two species ( see Neigel and A.vise 1986 for the exact g·enetic distance inequalitie.s}.. Based on. t.he r-e:levant mtDNA genetic dititances be,tween the individua.ls I surve,yed, the Aechmopborus color phases ex·hibit a polyphyletic relationship .. Alt.hou.gh the length of time that two daughte.r species will ,t~emain polyphyle:tic is de,pendent on. the demographic mode of speciation, it gene.rally can be expected to last, with high probability,, for at least N generations following initial isolation, where N is the: carrying capaci.ty of females in e.ach daughtet'· po,pulation (Neigel and J\vise 1986). The evolutionary effective. population size of females ( Nr".,) can be used to approximate H (Avise et al. 1988}. I estlmated NH*'' fo,r the Ae-c:h.mophorus grebes to be about 17, 800 female·s ( see below;: assum.e,5 an average gene.ration time of five years, as discussed in Materials and Methods)* 62 ".rhus, if the number of females that founded each of the color-phase populations: was r·oughly 8,.900, tbe polyphyletic relationship between them would be expected to las.t for a.t .least 8,900 gene.rations (ca. 44,500 years) after the .initial split.- If the propo,sed timing of Feerer.,s vi.carian.ce: model is correct (i.e .. , 6,000 yr BP) th.en the currently observed polyphyletic pattern is not s.urprisi.ng; there merely bas been insufficient time for the. development of a para-. or monophyletic pattern. A possible alternative to this "recent vicariance hypothesis 0 is one that involves, the. sec:ondax:y ex.change of mtDNA through hybridization and intr·ogression. Once two species reach a stage of monophyly, reestablisbme,nt of a polyphyletic pattern can only be achieved through secondary exchange of mtDMA (Meigel and Avis,e 1986). Could the currently obs.erved stage of polyphyly in the Aecl>mopho.rus grebes be the result of introgressive hybridization? Did the color-phase populations: actually become is.olated much farther· back in time than 6,000 years ago, reach a. stage of reclprocal monophyly during a long period of isolation, and then become polyph)lletic again after secondary contact? I find 1t unlikely because asyrruuetrical introgression of mtDNA would have to be· invoked to, e.xplain the observed high degree of polyphyly, and such genetic introgression between the color phases seems highly improbable given the observations tht1t today:. ( 1} both male occidentalis X f 1emale clarkii and 63 male clarkii X female occident-alis pairings (Jive rise to, hybrids ( Eichhorst pers,.., obaerv·.} i ( 2} int~:1J:mediate adults (if· e", presumed bybrlds} are present in m~ny b·.·'"'ee:ding populations {Feerer 1971', Rat.ti 197'9, De S~Ht 1.)87,, Eichhorst. and P'ar:kin 1991,. Eichhorst pe,r·s .. ot..:ze,rv .. ); and ( 3} female intermediates a:re fe:r·tlle (Nuechterlein 1981a, Eichhorst pers ... observ.) .. Genetic diveirgence between the· color ,pbases.--·I detected little or no mtDMA di.vergence between the Aechmophorus color phases. None of the 16 restriction enzymes that I e.mployed produced fragment patterns that clearly distinguished all specimens of oacidentalis from all those of claz'.ki.i ( '!'able 3) ... The 21 additional enzymes screened in my preliminary survey ( see Methods and Mate.rials above) llkewis.e did not produce any apparent spe:cie,s- specif ic fragment patterns. This absence of observable diagnostic mtDNA markers is re:flecte·d in the estimated low level of mtDNA divergence betw~en occiden·talis and clarkii (0 .. 19t when int:raspecific polymorphism is not accounted for, and e-ssenti.ally zero when it. is accounted fo.r), a level of divergence which is not only less ·than most of the mtDNA distance estimates curre,ntly available for othe,r closely related species: pairs of Nort,h American birds, but also less than estimates; for se'.veral subspecies pairs (Table 14.). Since the color phases appea.red to represent very recently sepa.rated po1pulations., as suggested by their 64 Table 14 ~· E,stimated rntDHA genetic distances ( percent nucleotide sequence divergence) reported for pairs of closely rEdated Morth American a.vian taxa. .. Since fe\Yi of the s:ources. cited below, calculated net divergence (dAJ, all percentages thus listed are est.imates of average divergence { dn) •. Taxon pair %. Source• Species Limnodromus scolopaceus-L. gri.seus 8.20 1 [)arus atricapill.us-P. oarolinensi.s 4.00 2 Quisoalus major-Q .. mexicamus 1 .. 60 1 Ra'!.lus elega11s-.R. longirostris 0 .. 60 1 Aechmophorus occ.identalis-A. clark.ii 0.19 3 Zonotrichia atricapi.l.la-z*. leucophrys 0 .. 11 4 Carduelis fla1nmea-c. horne.manni 0.16 10 Subspecies Pa.rus inornatus ·t.ra11sposi.tus-P .. i. rJdgwayi 5.02 5 P. wollweberi wollweberi-P., w. phil,lipsi 0.56 5 P- b.icolor bicolor,-P .. b. atricris'tat:us o •. ss 5 P.. gambeli gambeli·-P.. g· • .baileyi 2. .. 60 6 P. sclateri eldos-P .. s~ rayi 1. 40 6 [)asserculus sandwichensis-·P. s. rostratus 1.70 7 Geothlypis t:richas arizel.a-G. t· .. typhicola 1.20 8 G. t.. ari.zela-G," t. brach.idactylus 1.20 8 Pipilo erythrophthalmus .rLley:i-P. e .. curtatus 0.80 8 Branta oanadensis mi11ima-B. c. maxi,ma. L.10 9 B. c. mi11ima-B. c .. mof:fitti 0.80 9 B. c. mof£Ltti-B. a. 1naxima 0.26 9 Carduelis fla-nmre·a flammea--c .. f. rostra·ta 0.17 10 c. f. f.-c. f,. aa.baret 0 .. 19 10 C. f. rost.z:·ata-C .. t'. cabaret 0.26 10 ----·------·-- ~ (1) Avise and Zink 1988i (2) Mack et al. 1986; (3) this study; (4) Zink et al. 1991b; (5) Gill and Slikas 1992; (6) Gill et al~ 1993; (7) Zink et al. 1991a: (8) Ball and Avise 1992; (9) Shields and Wilson 1987b; (10) Seutin 1990. polyphyletic pattern, I attempted to estimate the amount of net sequence divergence between them.. However,. the resolving power of my mtDNA a.nalysis was not sufficient to 65 accurately detect a time of separation dis:tinguisbable from zero ti:me. before present ( s.ee Results above}., tlevertheless, since no fixed fragment-pattern differences r.,r,ere detected between the color phases, and the phylogenetic pattern between them was po,lyphyletic ,, I do, not reject Feerer' s vicariance model--that the Aecbmophorus color phases arose sometime within about the last 6,000 years. Clearly, additional surveys of Aechmophorus mt.DNA variation will be necessary to accurately assess the true level of nucleotide divergence between the color phases" Direct sequencing of the fastest evolving regions of the mtDNA. genome (e .. g., segment I and III of the control reg.ion; Quinn 1992, Wenink et al. 1994) could provide the nec:e·ssary re.solution to fully test the proposed timing of Feerer's vicariance scenario. The level of dlvergence between the nuclear-DNA genomes of the Aechmophorus color phases has been assessed. Ahlquist et al. (1987) and Bledsoe and Sheldon (1989a) used the technique of D'NA.-DNA hybridization to mea.sure the divergence between the sing·le-copy nuclear· DNA { scnDNA) sequences of occidentalis and clarki.i. The reliability of the n~ported levels of DNA-DNA hybridization dissimilarity (mean delta T!oH = 0.57, Ahlquist et al. 1987; mean delta mode;:;; 0.82, Bledsoe and Sheldon 1989a; see Bledsoe and Sheldon 1989b for discussion on co.rrelation between delta ii\0 H and delta :mode) has been questioned (see Chapter II) But if the estimates are reasonably accurate, they suggest a 66 level o·f nuc.lear-DMA divergence :of at least O .. 90% be-tween the two color phases (Ta.ble 15')'" In various ve:rtebrate cups,, including birds, it has been estimated that the mtDNP. genome evolve:s .5 to 10 times faster than the nuc:lear-DH:l:t genome (Brown et al ... 1979, Wilson. et al. 1985, Belm-Bychowsk.i 1984, Shields and Wilson 1987a; but see Vawter an Brown 1.98,6, Lynch a.nd Jarrell 19 9 3, Ramire:z. et al. 1 9 3s) • Thus, by extrapolation, if: the: nuclear-DNA genomes of the color phases have diverged by at least 0 .. 90\, their mtDNA. genomes could have di.ffered by 4.8%, at least. As discussed above, I clearly did not find such a level of mtDNA. di.vergence.. Such an extrapolation, however, is based on the a.ssumption that hybridiz.ation has not occurred between the color-phase popu.lations in the past. Such an assumption is unreasonable g·iven that hybridization does occur today. Tc) date, there are few pai:rs of avian t.axa for which genetic-distance estimates have bee:n made for both mtDNA and scnDNA. But, of the distance values that are available ( 11 able 15) , those for· the Tl1fted Titmouse pair ( Pa.rus L,icolor bicolo.r: versus P .. b .. atricristatus) are the most similar (id~:. ,. n.rtio of mtDNA distance to scnDNA distance} to the values fo,r the Aechmophorus· pa.i:r., These titmice are hybridizing semis.pecies (Mayr and Short 1970, AOU 1983) which interbreed freely in a narrow cont.act zone through east-central T·exas ( Dixon 1955) .. The tufted titmice and the. Tabl.e 15.. Comparison of genetic divergence values reported for pairs of North American avian taxa .. DNA-hybridization values {delta TM) for Parus ta:xa are from Sheldon et al. { 1992). Delta TM value for Aechmophorus taxa derived from Ts0R v~due (using equation l of Sheldon et al. 1992) reported by Ahlquist et al'b (19S7). scnDNA estimates based on 1:1.7 relationship between delta TM and percent nucleotide divergence (Cacc0ne et aL. 1988). Parus mtDNA divergence estimates (from inferred restriction-site data) are from Gill and Slikas (1992) and Gill et aL ( 1993) ~ Percent nucleotide d.iver';)'ence Taxon pair Delta TM sen.DNA mtDNA mtDNAiscnDNA Par:us 2.20 3.74 9.81 2~62 bicolor-P* wollweber.i ~ P~ bicolor-P~ inor:natus L.35 2 .• 29 7.,42 3~24 -.,,,,1 P~ b. bicolor-P. b~ atricristatus 0. 56tt 0 .. 95 0.55 0.58 Pt; carolinensis-P. atricapillus 0.42 0.71 4@60 6.48 P. car:olinensis-P. hud11onicus 0!<99 1.68 3.90 2.32 AflJchmophor:us oacidentalis-A~ clarkii 0~53 0*90 0.19 0'121 11 · This distance estimate may be e.xaggerated (Sheldon et al!t 1992). 68 Aechmophorus grebes biorth s:how a mtDFBA divergence rate that is. slow relative to the divergence. rate of the:ir respective nuclear-OMA g-enomes. ( 15). Such a pa:ttern in the:se hybrid.iz.ing fclr:ms versus the patte:r·n observed in no:nhybxidiz,ing fo,:t»f.lls: ( e .. g"', .Parus bicolor ve.rsus P.,, wollweberi ,,r F'/1' b.i.col.or versus p., inorna'tus,. Tahle lS) i.s axpe;cted ( e, .. g ... , Perr:is, et al of 1983, Sbi.elds and Wilson 1987a). Intraspec.ific phylogeog.,:~aphy and' female-med.iat.ed ,g,e·.ne flow i.n Aechmopborus. --·The irrtraspec.ific mtDMA ph:ylogen:y of .Aechmophorus' consists closely :related. haplotype:s {:Pig .. Sb) which appear· to be geographically widesp:read within the United States-C'.it'ln.ada. bre;edlng· r·i1tnc;Je ( Table 5) • O·f' ·th.e eigh't. mtD1'1.A genoty,Pes tha.t were dert.ecte.d in. m.ore. than a single specimen, seven of them represent haplotypes that were found in both the was.tern ( SW) and east,ern (l~E) ext:remes of the range ( Table 5) ~ Geo,graph.ic populations o:f ftpe,cies exhibiting this particular pattern of intraspecific phylogeograp.hy { i..-e", c:atego,ry IV of Avi.se e,'t al., 1987) are con~dde.red to, have had l"'elativel:t ex.tensive and re,cen't hia;to:rical lnter·cc.mnections ·thro,ugb gen.e f loiw ( A:vise et al ... 1987). In the case of the Aea.hmophorus grebes, two obs:ervations provide support. ·fo:r thi.s. thesis.., Firs:t,. c:onte.mp,oracy gene: flow among Aec:hmopho.rus breeding are1as within t.he. United States and Canada appears to be :mode.rate to h.igh { see: Chapter II} • The ephe·meral (5;91 fll«ad!:11i1:ei it.he Jl.aiJlt~;e:/cmiarrd~ habfi.itartt.ra; 1r·e:q1U1.:il.1red b,y Aec:Jbi/$JJa,Plln:Nrrai:,. fll"Ji:r ne:r§.t Second,. tJ111t:~i B:)01prtra.:JLati.01n ge:tue'lt;.;Jic: ~·.a:r·.:ila:ft.lrt:1,ni c:f' r:ttrt4J)nA obtH?t'.-Vt2Nl in :artt.11:voy· ~a!Jfi :t\td.atJL'<:Qe]ty l'°'~ lit~.dl 001 geog.r-aphic ~truc:tu1ri1~rJ of -t.hei vtair·iatJLc11ll waar. d.ot:eobid,.,, 'A 1 valtie of O.., 2J was: c;al~·ul&rted f(liJ;· tbei gi~oup CC!ff s;erveJ1! bi:t~tuid:ing s:ites.. Th.lB value m~:ans ·t.h~·t td1out, 231 of' the t.c,ttal in mt.D"1A :testric·tion-,si:te dlat;a die;tingu.. t.a;bad breedl 11119 and U11e c;.)rth.~:Jr.' 71111£) CO.(!C@:tred within groturp orf ind:ivdidiJJialo: t.J;atlffllplod :h:,c::111 tht'lse t.u:ee·dlng /i\Ji:t,11u11., .B;11.1t, sir:see o,ver 141; o:f; ·the :cando•l;y .... gene,rated. values ·wcire liu·::ger, tban. ·the caJl..cu.la;t.ed va.lue11 signif i.cant gec,graphlc strltc:tiu:.ing wa.s J'.Mlrlt, indic;a't,ed by the itnal-ysis ~ N10VA an.olya:is of: the ·t~,~o ge'.ographic.ally dlstnnt grcm.p$ breeding sit.es: ( Stir verstllS' :~E:;; Fi.g.. 4) pa rt it i.oned re a: t rict ion-site 11ra:riltntr0~· irrto about 981 fo,:r w1 thi'f1-br,,,eding si.t£.ui compone.:rrt. Likewls,e, t.he. AMOVA atU\lys,is: did not de,tect any a:lgn:ificant 9eograpblc ~tructtu.:-ing.., Under a fi.rd,te· i~land model {Wr.ight, 1943), the effective mig.r«11tion :r·ate ( N~m) be,t:weEUl popul.atlons can be 0 .. Si { l / Gs-11, -· 1 ) (Takahata and Pa.h:rmbi 1985}- tlhen N,;m is gre·atex: than LO,,, 7/({J) t l ., ·71; ge:©)gr:lt'·~phlc :tt:}egi.otHf., l I/; 7/, p,~--~rmi.o,t·ypies t intte:r-val/1Ji, (Cit)fillld n(O)t, be dact.e::tr'Wilin.ed :b,·t~' 'lt,he 141 b111dt th.~ la:1t·4Jaiudt, r·andc;ffl!ly-,gen1e:tatt:.ed. O.wJ;1· 1 ga:ve attt 'II~ '@ltl.ltU!e >·l c;, C "' ! b1t!j" f:ro;.~ tltia the· bypart.bermc!.11 ·t.bart, ge;fll4!1 flc,w wlthin th~ Aa:ch.MiophOJ,r'i/JtflJJ pc,p;!Ui.JLa,:tt.Ji.A,1:rii ~,ui blgh.., 11:t:toct:Jv• populafJ:l,011 •1•• ol!' Al8·etbmo;phorus f11,aa;J•11 ~ C•·-·tJ~ln(J .inb1I)tt~1dLb».g 'lt.huo,·ry Ii.Ht applied ·tc ne1u.·t:.r·al [email protected] mot.e:r'flally t A~.il'-ct ert, aJL. ~ «1988) ~iade ·the po,p1uilartio,n eJL2:e of #c the Red-wlnged cm.i. «11,·t:iffla·te o:f N1r1/fi 11 :f:r.··os cbS1€tr.ved a-nm:ogg rarndo1m p.)~:JL.r.:9 o.t 01:f l(ii w1Cf1(D) ~aie: S4S1-:f0Jld a11vualller· tb!it 2(01 m1.iJLl.io>'n fe:miale· l,1Jik.ewi$0, in ·two. coit.:ber h:igh-gene· f 't;,he,7 foitarnd th.art esrt.imiat,u, ai:f llrft•» 0if .ffltag1uitJJ.l.de ti~JUe;J~ 'It.ban pres;ent-· 11 avian :re~U1lt,tI, inr:'ltt:lld~: «1) the t~arte: of: m;tDNA co,:umii!Jlll011ily' clted blrd® ( 21 1,,,n'. 1m1i.lllon, yea.rs) ;JL,a; inc:ox·:rcect., ( 2) ·t.here ha.s p,oidt i \re di.rec:t:iona.1 :a.e,leci':ion. on :m'ltll.GA exp1.u1sion of feJnl'.ale p1oip)u.lat.icns (Av·i.at:ll et. a]. ., l9J88, Avl:s;e 1999, Avi~e; J.uuj Sall 1991) ,,, 11f'i1e last exp.lana't. .ion ]nltay for much of the oibse:rved disparity tr srbice mo,sdt. of the ·taxa surveyed to da.t.e ( e,. g.., , R.ed-·winged .Blackbird., Downy w<,caoec:.1tte:r, Common Cb;·a.ckle I_ Quls·calus qu:1sav:d.a], rodpo:l just 14·,000 becau.ae of 'the Wis:consin gla.cia·t.. lori l genetic di$tanee· bet.wee:n 54 .randomly-d_r·awn pairs o:f :t ndividua ln was Ofe JL 1 s:, y.ieldi.ng an lltr("'restima·te of 17, 800 f l Aeoiunophor:us grebes with.in the United State·s-Canada :cange n.iay f lu(';t,.u~te betwe·en about:. 6,0, 000 to l.00, 000 ( see C.bap·t.e1: II)~ The magnitude tld.s: discrepancy ( oinl y about six-, s;ted above, the Aec:hmo,pborus breeding range tod?1y is: large.r· than it was: during the: last. glacial period 12 tt11ff"l/"Will'"'·M~·ll"'t ·ltllru.Iti:Jk·e many oif tJ11e aborve t.axa 11• the Aec·bmopho:rtts porp1t1J.lat.io1n obvio1U1s:ly did n.o,t. expand. g1~eatly in size du1ting the last 1.4:,, (H)O yetn7s. The s:maillex· ,Nr·to-·Nffftdl:» ratio as .~uc:b,. 1:ndic:at.es that, "thei n:n:»mber of .Aechmopbo:rus has bee:n re:'.lLativ·ely· st.able over· tbe :r.·ocent evol uti.o.nax·y past.,, Evol utJ, ona.r:y and ta:Jt:onom1c i111pl:l..cat~J..oas. -·-My ras:t:t""iction-cn:zyme a.nalysie; does: not provide: a complete ·test of the null h:ypot.he2ds--A. oc:cident:al1s and A... clark1i are genetically indistinct.--.fo.r t.wo :r·eascris"' F'l:rs·t, only about 2, Vi: c,f the mtD!lA ge:n1ome was a.ssayed by t.he: 16 a:nz,ymes. Secondly, the mit.och.ondr·ial ge:no.m.e repre.s:en'ts only one of many ·~gene~~ gane,alog·ies withi.n tho Aecbmo_phorus pedigree"' i:onethele·ss t considering the more :rapid pace of m.t.DHA evolut.ion, rela't.ive, to 'the nuc:lea.r-DNA genome, and the de_monstrated abi.lity of compiurable· 1.u.tsays in .reveal.i.ng fixed mtDNA differe·m::e:s; between otJ1e;r closely related avian taxa ( e,g. t M~ck et al-. 1986,. A'Vi.se, a.nd Zi.nk 1988, Zink e't al. 1991b, Gill iil ,. 1993) t the fact t.hat~ no,t a slngle fixed f sts lit , .i r any fl genet.ic occurr(~d between the ./!usm·hmophorus color pbas.ers,., The color phi11ises a:rte, currently· cons.idered. to be ,.tbiolagical s1pacias;f-~ (AOU 198.5)., My flndings. do not directly bear upon the question of their status as biological species, which is defined on the basis of the 1.3 gene t 1c~ u1vergence....,jj. differenceg. had be,en. detected,., tbe cur.rent :ranking of 'the color ph~uH.?s as s:pe:4::d.e.s: would have been suppor·-ted. Conversely, however;: the abse.n.ce of debict.able: mt.D11A differences not ev·idence. for conspecificity. My· results only indicate that there has been considerable historical connectedness between the color phases ove:r· :recent evolutionary time. Whi.le1 the mtDMA results provide only equivoca.l support of the proposed timing o:f the vicariance event hypothesized in Peerer' s spec.iation model, conv,ersely the results clearly do not pr·ov:i.de evidence fox· the 1.7eje.ct.i.on of bis model. If the currently obse.rved mo:rphological and behav.ioral dlf'ftu:ence·s be·t.we.e,n the color phases dld arise with.in about the last G, 000 years, then those: changes have come about relatively rapidly. For this study I did not. oibtain any .individuals of 'the. two Mex:ican subspe,cie.s... Feere:r ( 19·77) suggested that 1n Me:x:.ico, :relative to t:hei.r northern counterparts: ~ m.a:y be. more sedentary beca.us.e the nef;tin.g season in M~xico is longer than the nesting season in the United States and Canada.!t :F'eerer ( 1977) also suggested that the level of hybridization between the color phase,s may be higher in the Mexican po1pula.tions ,. as evidenced by his di.scover1i that proportionately more intermediate-·:plumaged 74 ind.tvi.duals occu:r in the Mexican populations than in. po·pulations in ·the United States and Canada.. It is not known: if there is an~/ movement of gx:·ebes between breeding areas· in Mex.ico and the Uni.ted States (Ei.ohhors·t 1992). If not, then there po1t.entially could. be mtDMA. differences between the Mexican .Aecbmophorus populations and the United States-Canada populations., 1''1tu:c·e studies of genetic variation in the Aechmophorus grebes should include speo.imens, of both color phases, from the Mexica.n. populations. CHAPTER. J.V A REVIEW OF' SPECIATI.011 J(ODELS PR.OPOSSD FOR TBE WESTERR-·CLARK., S GRSSB COMPLEX (AB.CHKOPBORUS) In:troduction Climatic changes associated with. alternating Quaternary glacial and interglacial pe.rlods hav·e lmdoubtably promoted speciation in the avifauna of North America. Specia.tion models have bee.n presented for many Nort.h American avian taxa { Rand 194 8, Mengel 1964, 197'0, Se·lander 1965, Hubbard 197 3) • Bas.ically, these models are variations on a two--step theme: first, an ancestral species becomes continuously distributed over a wide area durlng· an interglacial period; second, during the following glacial period the species is :fragmented into two or more isolates by g·l.acial event 1 h Grtch ifrnlate di ffe t:;uliating in turn (Hubbard 1973).. This splitting of the. range of an ancestral species into two or more geographical isolates is commonly termed a vicariance event. 'lwo divergent speci.ation models have been proposed to explain the evolution of the two color phases in the Western Grebe complex (.Aechmophorus}.. The two color phases are currently recognized as separate spe.cies ,. A. occidental is 15 16 ( i .. ef', "'da:rk-pbase"I! :for:m) and A .. , c.1.arJdi ('. i .. e.,, ,mli.ght phase'~ form; AOU 1985), and are restricted in distribution to western North America. ( Storer and ·H'uechterlein 1992). One model, proposed by Ratti (1977), follows the a.hove two step theme, with a vicariance ev,e:nt being hypothesized during a glacial period. The other model, proposed by Feerer ( 1977), alternativ·ely invokes a v·i.cariance event during an interglacial period. These two models also differ with regard to their hypothe.ses about the relative geographic origins of the two color phases; an. east-west scenario ( Ratt ·1 1 7 7) versus a north-south scenario ( Feerer 1977). Neither of the specia.tion models for Aechmophorus has been critically reviewed. Here, I re.view these speciation models in light of: (1) current understanding about the glacial and climatic history of western North America during the Quaterna.ry Period; ( 2) the spatial and temporal distribution of Aechmophorus fossils; (3) the breeding· and wintering distribution of Aechmophorus; and (4) the nature of Aechmophorus migratory :movements. I show, from a biogeographic perspective, that Ratti's vicariance model is more unlikely than Feerer's vicariance model. Quaternary Hi.story of Western North America rrhis brief summary of Quaternary history is intended to provide the reader with selected information that is relevant to western North .America and to the Aechmophorus 11 :speciation models discussed in tbis ch.ap·ter+' For :more comprehensive and detailed treatises on 'North Am.erican Quaternary glacial, climatic, and ecolog·ical t.or1ics I refer the reader to Wright. ( 1983a, b), B:ryan.t. an.d Holloway ( 1985), Ruddiman and Wright ( :1981), Fu.lton ( 1989) ,, Pielou ( 1991), Clark and Lea (1992), and Dawson (1992) .. The Quaternary Period i.s subdivided into two epochs- the Pleistocene (ca .. 1 .. 65 million.-10,000 yr BP) and the Holocene (the past 10,000 years; Richmond and Fullerton 1986a). '.rhe Pleistocene epoch wa.s characterized by successive glacial-interglacial cycles; each cold period of glaciation was followed by a warmer interglacial period in which temperatures were generally as ~igh, or higher than those of the present Holoce:ne· interglacial., Deposits of at least 10 Pleistocene glaciations have been identified in mountain ranges in the western United States, including the Cascade and Sierra Nevada ranges (Easterbrook 1986, Fullerton 1986, Richmond and Fullerton 1986b). ~'he ice sheets of the Wisconsin glaciation reached their maximum extent about 20,000 to 18,000 yr BP, covering nearly all of Canada and the northern tier of the United States with ice ns much as 3.5 km thick {Pielou 1991). Soon thereafter the transition from glaciation to deg'laciation began as a result of increasing solar insolation (Milankovitch orbital forcing, Berger 1981). By 14,000 yr BP the .ice sheets were in full retreat .. Summer in.so-lation 78 in the Morthern H.emisphere peaked betwe:e·n. 11,000 and 10,,000 yr BP, and by 9, 000 yr BP muich o:f Canada west of longitude 100~1 was ice free ( Dyke and P:t'eis:t 1987}. The we'.f;;te.rn region of the Un..i.ted Sta.tea south to central Mexicc,. co,nt.ains n.111m.erous hydrologically closed basins, only some of which ,.n:.1rrently contain shallow ephemeral lakes.., During· the Late Wisconsin (ca. 35,000- 10, 000 ·J ~ :EIP), however, many if not most of these basins contained some l~kes of subs·tantial size, common.ly r.'efer.red to as "pluvial lakes"- ( Smith and Stre;e.t-Per,rot:t 1983, Ben.son and Thompson 1981) .. The largest group of pluvial lakes was within the Great Basin region O·f C'-=lli.fornia, 'N'evada, Utah, and Oregon ( see F.ig. 7) ;. others. e;xisted i.n parts. of Arizona, New Mexico, and northern Mexico. Late-Wisconsin expansion of pluvial lakes in the western Uni.t.ed States occurred during the period .from about 2.5, 000 to 10,000 yr BP, with maximum levels being reached by about 15,000 yr BP (:Smith and Street-Perrott 1983, Benson a.nd Thompson 1987) •. Between 10,000 and 5,000 yr BP ma.ny areas i.n the wes·tern United States apparently were characterized by drought, which culminated between 6,000 and 5,000 yr BP, when many lakes stood at h)wer than today's, or were dry ( Smith and Street-Perrott 1983, Wehb 1985). Reexpansion of some lakes, especially in the western margins of the Great Basin, has occurred since 5:, 000 y:r. BP { Smith and Street-Perrott 1983)" Much less is know about the Great Basin lakes during- the 19 Figure 7" Map showing extent of 'Late Wisconsin glaciers in the Cascade and Slerra Ne1vada ranges, and pluvial lakes within the Great .Basin, wester.·n United Sta,tes. Map was compiled and modified from maps in Porter et a.l ... ( 1983) and Willia.ms and Bedi.ng·er' ( 1984,}"' (>; AN 1.1 110""' [ffi1ffi]j Cordmen:u, ... Laun·ntide ke· s:hetl'I Cascade· ond Sierra Nev,ad:a gJaders; • PtuvfoJ lakes 81 J:1i,1td.le'. ar1;dJ Earl.Y Wlis,,~onairn but t.lliey .lik.e:ly were more: t1'.Xt~ru,ive, tha.fll art fBLr'.llchh1u1be:t' 1992'., o.-~iartt;tt, and. Mc:Coy l 9 92) .. L,ut.'ge fJil 1Uvia.l s;y~·t.e~tooS{ miay, ba:~e: cM:::cui:1r1'.·ed do:ring· previous glacial advances:, pos;si:ibly at: 1010.., Oi0(11-·yeer int.(:::rval~ (BentH)n and Th.o:mpscm. 1981) 11, The H,.,,loce:ru? clitnalte of J4lox:·tb Amexlca,, as: inferred fr·ctm the f oiH;il pollen re·co,rd t ge:ne1tally has been charaott.a.rJLzed dry mid-JJolcce:~:oe; and ( J) a cool-moi,9tt lat,e-9'olocene ( Bryant and Holloway 1985)" ~r·he mid-Bolocf!ute wa:rm-dcy climatic has gone under· va,rioua n.ame-s, such as Al:titbermal, Climatic: Clptlmumt Hypsithe.:rJ~td'.,,. and Xerotihermic11r Hypsithermal ( Oeeve~l !"lint l9S.1) i~ now li.Ufe:d a]ww1at uni var ly for Horth e:.x.cept. in the ~,eat whe:re Altithermal ( Antet;s; 1948, ) .is mo:rt,, ;fre,,3uEn1'tly used. Aechmopbo:rua1 Die,tr,ibutio,n and Migr,a"tion 'rhe breeding r'ange of .Aechm·opho.rus 9:t"ebe,s ls ~.hown in 2: { 1.H1ie: Cbapt.er II). B·:re.e:ding occurs a't widely su e lakes/marshes within this range ( tl 41: 9 •. , Koon4! a Rakm~s11d 1985, ,. Campbell et al.. 199011 Andr.QW·~: and Rightett.: 19 ,, Semenc1huk 1992)"' Lakes/marslles p t:'€lV ic11U rJies,,ting can beet1me1 un.sui table·, nlly du.ring of drought ( Eichhorst and Park.in 1 !J91,, E:ichhoirs:t "'· obse:rv ., ) ., Since iUH.:::hmophorus grebEHJ: wint.e,r on the Pa.cit: l l) , but breed at sit.es; ht1:odl.reds; 'to 82: __ ,,_,, ____ ,,g,_.,,, ·ttJhier 91e.n11:n,al d:Jt.Jr·eictiottll o:l[ ~·tf:Jiertt;.'{lw1ai,:ird .lLn fail Jl..; brut. biuided CJi:tu tb.e a.ame M!anddt.co:;ba breedLlLn.g 9:1iib11Jcquerxrtly· reoorvei.r,ed ·tJ,a:t ,1:'in:ter ~e:r·at .f:ound art, mt:lt,«ui Jin ~,~shin.gt,,1:fi,. ©it(;~?: ~Ji~~: f (jJiUJtlntl! :in ,tt,Grm:itr·al C:~lifo1t:n.ti,~, a.~d. a 1ne: was, (C'.(i)Jliformria «!Sle•(i] E:lir,J(lb wn i1J,it:<1.1ina: in 9Jlia1.cd,alL. jpte.r lad. f .~ecbtrriti0Jpbcrc'fklf 1Et a. mH,J»Mib~r a,f ·ft.be JJil~vial rtortb ,,, :eo111ea; 01f: Aoc/1-opbla>.rus of :tt,a,to,,..,W/i.tD:a(MUJ.:ini a~Jc (( .,.~.,,.,~Y=·='·i' ~.c~ltt~i!o:f Cave; su?,e :t1lgi •. ll. ln «f'o:;u::t, R the Pa(:i·fJLc coa&'1t 11, exce;ep1t tJ'll.at 'the l hr0undaJey l\!41.y h~vo ab10ut. Jl~'C'1:Mlff.iaf)1horrfll1J;' .ba:ve beeri fou:nd. .in ~ge [email protected]€,d arl /0)~9 'the «~oe Fig. l in II). Spctt*!1:1.ation Hod'.•l• « l ) W ~'.Jf~:Cl,g r r~rpJ1.i c-1 al ~ tbe, li 9hdt-J>iha$e ~ ~Ii:f)1Uth11J;>rJTU 8!) IJ'OP11Uilatic1n"' { 1965) ,. boV*Pe:~;rer, d:id rMJi'ft sro.J.ggest. a geogrcJ1p:hic ba.rrie11r,;, 011:· on whefui the v.icair.iance ~-v~n,t ©c:c::u:r r~d ., nat;t.:i « Bri 1· » s:1W99~~t.ed. t.ba.t d.nrJLng U1e p, lei srto:eenie a a.ing,le pop1wla.tion. of Aecih.tnopbo.r.1u1 g:r·eb:es may have ,tx:i~rt.ed t:hrougbotudl:, C:ali:fo:tnia. 11 Ne·vad.a, aJ1.d Utah, with per.ip,her·al populatJ.o,n.s in Or·egonJ' Id«11bo,. Colo:r·ado, and '"'' t.ha.t g:rebe:s fro;ffl w·ersrter·n Cali:f'.o:rnia m~.y ha,·fe from those in Ne:vada and Utah as a r~:~tdt a. g.lacial arm ext.ei"Jidi:ng f 1romi Wlashingt.;on,, down Hery·ada. int.o 90,uU·u!'::t]ll California... While , divergence ligbt.- and dark-,1ihase f'o,r.ms. Ratt.i t.h~'? li.gbt·-pihaae forrm pais:sdbly develop,ed in ate, and e:xtended .i't,s range so,uthwa.rd, along the d:rainage ba.aint into Me~icN). The da:rk pha~G fo,rm in u,e wesb!rn isola:te and, with l:flCE.H3: S i Oi"f'l , tnirte:nded its range nuo:rt;hward to the Ir east: .into southern Canada., 'flbe Crw.une, into sie,condary contact a:fter the g;l~\C·ial R.attl ~ 191'1} also propoae,d a second vex1sion of his , wb.icrh he modi.:fied to be compatible with a hypt.1,t.h€!sis in which .i~ec'11Jmophorras gr·ebes were polymorphic (Mayr and Short !970) prior to speciation~ In this 84 scenario··, a large p,olymo1rphic population of Aec:hmopho:c:us 9:rebe~ be·«:~amf2 divide'dl 1:n'l!:o two is:ola.ted pop·01Jlat.io:n.s.. During iS'olati,)n,. natlhll:ral selectloir» el.imina:ted da:11,r.k.-pbase indiv idua.1~ fro,m Otlf;: po)pularti.ofll and 11ght-phase lndi.v·lduals fr,Jm -the otheir p,01pru.1ilart.lon.. tvlhe:n. t.be two popu.latlons came i.nto secondary contact. :n?.produc·tlve i.sol.a.t.ion was ma.i.nta.i.ned by isolating :mechanis;ms t.ha.t had develo,ped during isolat.io:n •. Feere·r { 1917) also su.ggest.ad a vica:r·iance Jf,odel for the Aec-hmophcn:us· grebes, but one which d1d not. invoke isolation chJt:·ing· a 1 period.. J\cco:rdir»g -to F·ee:r·er;; s scenario, these grebes: were geographically con·tinuous in disrt:r:ibut.i.on throughout the, Pleistocene, uti.lizlng 'the pluvial lakes in western North Al1ner.iaa~ Af·tex" tt1.ei W.isc:onsin gla.cia,l period, during 'the arid Hypsi·thermal ir,rte:rv,al o:f' the mid-,Holoce.ne, desic.cation of pluv.iail lakes in the wes,,te'.rn Un.i ted ef 'fectively isolated .Ae,chmophortas: grebes in the south from thoe:e in the n.orthl\' T1he, aourt.her·n and nor·thern segm.ents were potent.ially isolated for. up t.o 4,000 years, during whi.ch time: t.he light- and dax,k-phaee forms evolved ln the southern and northeirn populations, ,respectively.. At the end the at"id period, th~'?! ensuing we,t period :re-established bet\~'e ll'i.scu.ssi.on I Ratti s: models. -· ..... Both of: Ratt.i ff' s vicariance models glacial arm 85 extending f :rom1 ~Wash.ington, dloi11~.tr11 the Sierra :Hevada in·to so,u.thern Cal.i.fo:rnian, ( Ratti 1977: 122)"' Ra:tt.i II s gl.acial aruf is a :re:f er enc,~ to the s;ex"ies of .mou.iitaln glac.ie:r·s t.ha't. occurred along the Cascade Range /O)f 'Wlashi.ngto:n, Oregon., and California, and the Sierra He.vada Range of Ca.lifo:rn.ia,. during successive Pleistocene glaciati.ons.., The Cascade Range extends for l, 120 km from !~assen Peak in California ( latitude 4.0<61~) northwa:rds to sotl't.he·rn B:.c·it.i.sh Columbia beyond latitude, 49g/.N.. The Sierra Nevada Range extends for 690 km, from just. south of' the Cascade Range in Cali:fornia, southeastward t.o latitude 35Cl·JO ''N.. Contrary to, Ratti fl s claim, there was not a single, continuous Cascade-Sierra Nevada glac.ial arm (see Fig ... 7)"' In Ore;gon, a continuous system of glaciers mantled. the crest oft.he Cascade Range for ne·arly 270 km, f.rom near latitude 4S'°M to near latitude 4. 2~30 '"N ( Porter et al... 198.3) .. South o:f lati.tude 42ci,30 'N, a limited number of small,. isolated glaciers occurred in the California Ca (Porter et al. 1983)~ In the Sierra l~ev-a.da Range" a glaciErr sy·s'tem,. averaging about 50 km wide, extended S:1ome 4.50 km between .la·titude:s 40°·N and 36°H (Porter et al. 1983, Pullert.on 1986)., Ratti ( 1911} noted that his; hypotheses on how the two color phases e:vo,lved represented spe:culati.on in the absence of banding and other da.t.a. Given the information on Aechmop,t1orus distribution and migration provided in Chapter II and this chapter, I argue that the glaciated Cascade and 86 Nevada ittountain ranges could not ha.ve complet(ely prevented t.he :movement of grebes be·tween. the Great Basin and west.ern Ore:go:n and Californ.ia., Under the r,·easonable p.remise that gtJne flow would ha·ve occurred bet,r~reen breeding areas located o,n oppo,£dte sdd.~es: of the Cascade and Sierra. Nevada mo·untain ranges, i.f ther·e was e•ast-to-west and west- to-eaat moven,ent of grebes" I suggest. three· ways in. which such movement potentially could have occurred. First, grebes might. hav,e migrated over gl.aoi.ated mountain ter:t"ain.. The presence o:f mountain. glaciers is not prima-facie evidence that. Aechmophorus grebes were incapable of, or avoided flying over, such terrain. Today, given the high degree of mountainous terrain t.hat exists between the Pacific coast winte::ring areas and :inland br·eeding areas, Aechmophorus: grebes undoubtably do .fly over mounta.in ra.nges ( some individuals, may even fly over some of the 700+ glaciers that are presently located in the combined Cascade Sierra Nevada mountain region in the United States). It is conceivable then that sonte grebes, potentially migrated over glaciated mountain terrain during Pleistocene glacial periods. Secondt might have migrated over nonglaciated ffi.()unte1in terra1n that e:x.isted in no:rthern California { see E'"ig ~ 7} • Fo1.r those grebe:s that nested at lakes/marshes within. southeastern Oreg'Oin or northwestern Ne1vada, migrating over an unglaciated region, wes,tward to the Pac:if ic coast, 81 pres.um~bly would ha~:.re been energ,etically :much less de·mamdlirtig, and taken less ti:me than flyi.n.g directly south,. beyond ·the s:outhern end 01:f the Sierra. Hevada Range, to a much mo,re distant wintering site.,, Third, gre1bes m:lght have migra·ted ac.ross the region of Cal.ifor:n.ia directly sou.th of' the Sierra. Nevada Ra.nge ( see Pig .. 7)'" Regardless of whet.her or not any grebes migrated across, any re9ion ( s) along t,he: combined leng·t.h of the Cascade and Sierra Nevada :ranges, numer·ous .indi.viduals likely migrated across the region directly south of the Sierra Nevada Range. Feerer' s model .. -·-·Feere:r suggested tha.t. the ex:tensive; pluvial lake desiccation which occurred over most of the wei..;tern Unite:d St.ates during 'the Hypsithe.rmal interval could have eff/ectively isolated a sedentary population of iAeahmophorus grebes in Mexico from a second population that existed north of the de:siccated pluvial-lakes region. Sine,~ Feerer's original proposal of his model, there have been additional s.tudies of plu1via.l lake basins ( e .. g .. , Smith and Street-Perrott 1983, Benson and Thompson 1981', Forester 198'.7, Waters 1989)" In the most comprehensive study, Smith and Street-Perrott (1983) su:mmarized lake level information f.rc)m 31 radio.carbon dated basins located in the ~e.stern United States~ They found that during the last 30,000 , pluvial lakes w1ere at their lowest lev~':'ls (i.e., 0- 15% of maximum levels) betwe·en 6,000 and 5,.000 yr BP.. These 88 pluv.ial-lalk:~ dat~ ai:ice cong:ru.e:u:tt with Feerer..., s hypothesis -that the vili~ariance event occurred sometime around 6 ,. 000 yr BP (i .. e .. ,. the mid-Holocene)., Additionally, fossil pollen evidence front a,a:os,s; 'the western. Uni.ted St.ates, .indicates that without quali.f:ication th.e, mid-Holocene period was exceptionally warm and dry (Brya.nt. and Holloway· 19'8,5)., Conclusi.ons Based on a bi.ogeographic perspective, I have shown that Ratti, s v icariance mc,del is more unlikely than F'eerer' s vicariance model .. Feerer's model remains a reasonable hypothesis given the available evidence on fluctuat.lons in. pluvial-lake levels .. As I discussed in Cha.pter III, the, :results i:rom the mtDNA analysis also provides information that is relevant to Feerer's proposed model. While the mtDNA results provide only equivocal support o:f the propoaed timing of the vicariance event hypothesized in Feerer's specia.tion model11 conversely the results clearly do not, provide ev,idence for rejection of his model., CHAPTER V RECOMMEHDAT.'IONS FOR. FUTURE RESEARCH ou· n:s B'VOLUTIONARY HIS/:fOR!' OF TBB ABCBMOPBORUS GREBES 'I'he purpose of this chapter' is 'to outllne some .ideas for future research proje,cts which I belie:ve· would help pr:ovide additional insight into: the e,.tolutionary history of the Aechmophorus: color phases.. The list pre,sented below is not meant to be exhaustive, and the idea.s outlined therein list are not given in any pa.rticular o:rder .. 1) Further te:st.ing of Feere:r s vicariance model wi.11 require additional estimates of the degree of genetic divergence between the Aechmophorus color phases (see also t3 below ) • As I mentioned in Ch.apter· III,. direct sequencing of the fas.test e'volving regions of' the rntDNA gEmome (, e .. g ~, segtMrnt I and III of the control region) potentially could provide the necessary re.solution to more fully test the proposed timing of f,"."ee:rer's vicariance scenario.., 2) Feert~r ir s mode:l could also be tested more directly through an archeofaunal survey .. Currently, limited 89 9(()) A,1,ch:mophoru.s fossil nr11ater ial is available frown Holocene-age deposits: { see Table 1 in Chapttu: II)*· Since not all pluvial lakes in the t,.;r,estern United States dried-up during the H.yp~ither.mal intet·val,. fossil !ln.11:rveys shoidd he condu,e·t.ed at basins that :retained water du:ri,ng th:is mid-Holocene period.., I£ A~r;;;hmP,phQ.TUH- fit}g.s,,ils we:re t.o. be founc:.l at iieveral. lake bas.ins, th~n Fe.erer • s inode.l would be ques-t.ionable ,,, 3) Surveys of nuclear-DNA variation within and bet.ween the Aeahmophorus color phases sh.ou.ld be conducted" As dis.cussed in Chapter II, 'the re:liabilit.y of the Aec:hmopbo:rns DNA-DNA hybr.idi za'ti.on data eet, has been questioned. The use: of the DNA-DNA hybridizatlon technique :for assessdng the: degree of nuclear-·DNA dive·rgence be:tween close.ly re,lated speci.es or populations is questionable," Othe:r- te·elmiques such as microsate·llit.e analysi,s would be mo:re appropriate .. 4} The mtDllA size varia·tion and indi·vidual heteroplasmy that I observed ln five species o:f grebes ( see Chapter J.-11) is o.f great interest since it has been observed in a limi·ted number of avian species~ It raises the question of whether or not this s:lze var:iat..ia,n occur·s in the mtDHA genomes of all grebe s;p,ecies.. If not, then the presence of the size variat1on would be phylogenetically lnformative. i~ limited ( gee Chapter II a,~r:i :EJ:1c'11lhor·st 1992). Such inf orm~1:tio,n can pi:rorvide a :nie~:-,s. of det.ermining the exten·t o·f contemporary gene flow bet.~een b:ree:d.ing a.:reas •. More banding· and color-marking of grebes s.uch as: What. ls; t.J11e ext..ent. of brEu:td.ing s:lte and wi.:nt:..e:r·ing philopatry? I's ·the:r,! move:me:n.t. of indiv.:iduals bet.ween Mexican and Uni.ted Stat.es; ..... canada b:r·eedlrig areas? Do the movememts of Clark., :s Grebes di.ft·e.r from 'the :m.ove:ments of Western Gre:bes? t.be level of hybridization be'twee:n the Aechmoplu::,rus colo.1~· phaa:es were do\ne solely by dete.rmining the relati.ve :f :r·equency o,f mixed-phase pairs; in breeding populations If F'ew det~ails ar·e- avai.lable about. the reproductive success of such mixed pairs, relative, to conspecifie: pa.ire:" A det.ailed study of mate choice in these grebes: is 1u1eded" What are the conditions u.nder· which hybridizatlcm will occmr?· ls the ove.rall level of hybridizati.on between t.he colo:r phases. incr,ea.sinq,. o:r· decreasing over time,? Peer.·Eu: (' 1971) sugge,s-ted t.hat the level h.ybri.dlzation nu.ty be: hlghe;r in the Mexican popu.laticH1s.. Is that trU.El" and if so, why? 7) Adults with intermediate bill-color and/or facial p11lt tern phenotypes ar·e1 pr-es.umed to be hybrids, but it has 92 y~t- to be co1trilf irmedl by raiis;imig known. hybrid! chick.s .. :?he·n,Ytyp,-£'. va.riart1o,:n o)f bybr-id\!$ :nie:eds to be stud.:iLed!., 8) Tlie~re i1l little inrfo:rma.tion concierniing t.bose. Aechmopborus grebe.a;, that. b.t:·eed 1n. .Mexico.. Jt. ia not even kno:wn lf· t.he, p,haa;e-srpe·cif:ic .Adve:rtisirv.g call cUf'f;er·ermes obs;,2.rved in thE!i Uini-ted States: and Ca:nad.ian 1,opula:t.ions ls true f'-.n: the J1f)1:xlcan pop1ulat.io1ru~;.., F'ec-,rer ( 19·71·» suggest.ed that Aec-Jimophorus· grebes in Mexico, relati've ·to their· northern oourlterpzu:-ts;, may be mor·e aeden't.axy... I'.f so, then there poterttially cou.ld. be; geru:z:·t.ic differences be·tween ·the Mexican and. the Unit,ed Sta.·tt:ui-C.a.nada p:o,pula:t:lons.. Fub;ic.re studios of varia.t.ion in the, JJechmopho.r-us grebes should include spec.:bne:ns of bot.h color pha,s:es :f r·o):m, the, Mexican populations. AP'P'EW1.0J:X ln,fe,rred mt1J:ti1' Re:a:t:r·.i.ctlon Sites W're~~nf.:.~/ rmia tt'~.:t· .i:)f,: of mtD1mi 1ceisdt.r.itYt;.i.o)ni. si·t.ers i :ni f;er,r·ed the :rE,5r{C; :t'lc.t.ion-eni.z.y• f r·ag~i:a:[t pat.bi;rr::ns oif th~: 109 gr~bes su.:tv·e-yeid. in t;hi.s; ai·tlilid:y· ... ·pre:ft':ene:~ ('~1tJ\p c;j,r, al,is;,tr.oce. «~oim) o:t: ea.c:b. si.tei jLs illldicartedl -fo:t (laeh 01:f t.h\(!; ]2 lhiJctip,lot:ypes. 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