INTER-ISLAND GENETIC AND CULTURAL VARIATION IN THE THICK-BLLED VIRE0 ( Vireo crassirostris)
Marlene Rona Waiker
A thesis subrnitted in confomity with the requirements for the degee of Master of Science Graduate Department of Zoology University of Toronto
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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protege cette thèse. thesis nor substantial extracts i?om it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permis sion. autorisation. NER-ISLAND GENETIC AND CULTURAL VARIATION
IN THE THICK-BILLED VIRE0
(Vire0 crassirosfris)
Master of Science 1998
Graduate Department of Zoology, University of Toronto
Marlene Rona Walker
ABSTRACT
Patterns of divergence in two evolutionary pathways, cultural and genetic, are assessed in the West
Indian Thick-bikd Vireo (Vireo crassiroslris). Spectrographie assay of songs of 140 males recorded
on eight islands formed three clusters: 'Cayman' cluster (Grand Cayman and Cayman Brac);
'Bahama' platfom cluster (Abaco, Andros and New Providence), and the 'trench' cluster - birds on islands separated by deep sea trenches (San Salvador and Turks and Caicos). Genetic variation was assessed by sequencing 389 bp of the control region, Domain 1 (mtDNA) which revealed 22 transitions arnong 17 haplotypes cornmensurate with recent speciation events, c. 1 10,000 ybp. The
Turks and Caicos and Cayman populations show genetic and memetic differentiation, in contrat to the northern Bahamas which are characterized by the mixing of thick-bill populations. This study has shown that geographic distance and isolation from potential colonization sources promote the activation of the micro-evolutionary processes which yield intraspecific variation. REPRODUCE AS IS
TO- WHOM IT MAY CONCERN:
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thesis wi17 be available at the University of Toronto.'~ Graduate Department of .
Signature Date ACKNOWLEDGMENTS
Speciai acknowledgment goes to Dr. Jon C. Barlow, rny supervisor, who has aiways believed in me. 1
thank him for providig never-ending support and guidance, and for sharing his wide-ranging
experience in ornithology, in particular, his extensive knowledge of vireos. In this regard, his
fieldwork spanning 30 years, including tape-recording and collecting in the Bahamas and in the
Cayrnan Islands, was essential to my current work.
When he first suggested that 1 investigate birdsong during undergraduate studies, 1 was filled with
trepidation. 1 loved birds, but it was the visual experience that thriiied me. 1 hadn't paid much attention
to the beauty of song, subtle contact notes, the delight of the Winter Wren in fiil1 morning Song or the
energetic bursts of early moming Song of the Northern Cardinal. in addition, I contended that 1 didn't
have an 'ear' for music, as evidenced by the traumatic experience when 1 was forced to 'mouth' the words in the Grade 2 choir. My next traumatic musical event occumed in Grade 6 when 1 was asked to
choose a musical instrument. I chose the trombone but was relegated to the flute instead. I disliked the sound of the flute for the next 30 years until I becarne mesmerized by the flute-Iike cadence of the
Wood Thnish. The study of birdsong has been a gift which 1 cherish thanks to Dr. Barlow.
Thank you to Dr. David Dunham, my adjunct supervisor, for his advice and encouragement since the beginning of my zoological 'adventure' and to Dr. James Rising for the frtscinating field experiences in Sape10 Island (Georgia) and Costa Rica, and for challenging me in the classroom. And to Dr. Allan
J. Baker, who taught the wonders of molecular evohtion, and whose constructive comments and suggestions in the course of my work have been appreciated.
iii Dr. Alejandro Lynch is gratefùlly acknowledged for his statisticd, cornputer and mathematical wizardry which he generously provided whenever he was asked.
Colette Baril has been a source of inspiration. We have worked alongside one another for two years, studying birdsong and 'discovering' that "evolution really does happen." We have compared syllables, read in nucleotide sequences, and shared the trials, tribulations and successes in molecular work. I am gratehl for dl of her help and I ch&& the time we have shared.
Oliver Haddrath has played a very important role in my graduate career. We took our graduate courses together, oflen collaborating in group projects in Conservation Biology. But it has been in the molecular laboratory that Oliver has been my 'rock'. He has always been cheerfil, patient and has helped me in so many ways. He has a gift at teaching, and I will always rernember his analogies, blackboard diagrams and spontaneous sketches of molecular evolutionary techniques and concepts. It is with a deep sense of gratitude that 1 acknowledge a vexy wonderful colleague and friend.
Mark Peck has been my fnend and field colleague in most of my fieldwork. We have traveled and worked together in New Providence, Andros, Abaco and San Salvador in the Bahamas, and in
Providenciales and Middle Caicos in the Turks and Caicos. No matter where we went his sense of humour,joyfiil interaction with people and his field expertise were remarkable. Mark demonstrates amazing tenacity and creativity in the field, and 1 wilI remember with fondness the many experiences we shared. I also wish to thank Mark for his help in the molecular laboratory and in the collections. There are rnany staff and fellow graduate students at the Centre for Biodiversity and Conservation
Biology at the Royal Ontario Museum whorn 1 wish to thank for their assistance, support and
camaraderie over the iast two years: Dilara Ally, Cathy Ayley, Maryann Burbidge, Alana Danko, Mike
Dennison, Judy Edwards-Davies, Andrew Given, Alessandro Grapputo, Annette Greenslade, Cortîand
Griswold, Brad MiIlen, Glenn Murphy, Chris Pankewycz, Tara Paton, Cathy Rutland and Nicola
Wade.
Many thanks to Dr. Sherwin S. Desser for his encouragement throughout my programme and to the
administrative staff in the Graduate Department of Zoology, Sheila Freeman, Elizabeth Tudor-
Mulroney and Susan Del Tufo.
1 wish to express my gratitude to the foflowing for financiai support received in the course of my
studies:
JC Barlow, Trust Fund (ROM), for my research in the moIecular and sound laboratories, for fieldwork in the Bahamas, Cayrnan Islands and Turks and Caicos Islands, and for conference fùnding for trips to
Aniba, and St. Louis, Missouri where 1 presented papers on the Thick-biIled Vireo; The Arnerican
Museum of Natural History and the Frank M. Chapman Mernorial Fund for the generous gift of research fiinds which supported iny molecular analysis; The University of Toronto which provided me with two Open Master's Fellowships; The University of Toronto, Department of Zoology for giving me conference hdsfor presentations at the Society of Caribbean Ornithology (1996, 1997) and for the North Amencan Oniithological Conference in St. Louis, Missouri (1998). In addition, 1 have met and liaised with so many wondefil people fiom al1 over the West Indies.
Thanks to:
Patricia Bradley who accommodated me in hm home on Grand Cayman, chauffeured me around the
islanà, traveleâ with me to Cayman Brac, assisted in the field and made numerous contacts for my
fieldwork in both the Cayman Islands and in the Turks and Caicos. 1 treasure the fnendship diat has
grown. Thanks also to Patricia's husbanâ, Michael Bradley for his hospitality.
Wallace Platts, Chairman of the Cayman Brac National Tmst who met Jon Barlow, Patricia Bradley
and myself at the Cayman Brac airport and thereupon acted as willing guide, and driver and assisted in
any way he could during ou fieldwork on the island.
Gina Ebanks-Petrie, Director of Environment, and the Department of the Environment for issuing a
research permit for my work in the Cayrnan Islands.
Arhuo Kirkcomell and Orlando Garrido, my Cuban fiiends, who have gladiy provided me with both
Song and tissue in order to andyze the Paredon Grande Cay population of the Thick-billed Vireo. Our
ongoing contact over the last two years has been a 'bonus' in my academic career.
Many people facilitated my fieldwork in the Bahamas. Thanks to:
Dr. Maurice Isaacs, Veterinary Officer and Eric Carey, Assistant Agicultural Officer fiorn the
Department of Agriculture for supplying me with a research pennit. Lynn Gape, Public Relations and
Education Officer of The Bahanas National Trust provided excellent contacts in the Bahamas. Among
vi these contacts were Anthony W. White and PericIes Maillis who gave me more contacts on each
island. In San Salvador, Mark and 1 stayed at the Bahamian Field Station where we were pennitted to
stay despite the station being closed. Velda Phelps shopped for us and generally made sure that we had
everything we needed.
Anthony W. White for reviewing his records of thick-bills in the Exumas and for sending recordings
of the Thick-billed Vireo, reçorded by Bruce Hallett on Providencides, Turks and Caicos Islands.
The Flo~idaMuseum of Naturd History for Thick-billed Vireo recordings by P. William Smith in
FIorida.
Judy Garland, Chief Conservation Officer and Ministry of Natural Resources, Department of
Environment and Coastal Resources for providing a researcb permit to do fieldwork in the Turks and
Caicos.
EthIyn Gibbs-Williams of the Turks and Caicos National Trust wlio was a welcoming face on
Providenciales. She joined Mark and 1 in the field directing us to good thick-bill habitat and was
instrumental in giving us contacts for our trip to Middle Caicos.
Dr. Michael T. Murphy (Department of Biology, Hartwick College, Oneonîa, New York 13820) for
his capture records of Thick-billed Vireos in San Salvador, Bahamas.
Special acknowledgrnent to my husband, Joel, and my children, Adam and Elysha, who have been patient, supporîive and encouraging while 1 have pursued this degree. TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES xiii
LIST OF APPENDICES
TNTRODUCTION
Cultural and genetic evolution
Profile of the species
Distribution and current taxonomy
A biogeographical perspective of the West Indies
Objectives
MATERIALS AND METHODS
Bioacoustic Analysis
Field equiprnent and method of recording
Recordhg parameters
Definitions
Sound laboratory andysis
Cluster analysis
MaWnum parsirnony analysis
Statistical anaiysis
Merging of datasets
Song type analysis
viii MATERIALS AND METHODS (Cont.)
Genetic Analysis
Collection of blood samples
Molecular laboratory analysis
Phylogenetic analysis
Statistical analysis
AnaJysis of Hunicane Data
RESULTS
Bioacoustic Results
Audiospectrographic analysis
Song type analysis
Genetic Results
Intraspeci fiç variability
Statistical analysis
Phenetic anaiysis of Vireo crussirosfris and congeneric vireos
Humcane Activity in the West Indjes DiSCUSSION
Song type variability consistent with islaud conditions
Cornparison of the songs of Vireo crassirosfrisand Vireo griseus
Hypotheses for wide-ranging distribution of Vireo crassirosfris
The impact of hurricanes
Origin of Cayman Thick-billed Vireos
Founder effect in the Turks and Caicos Islands
Coalescent theory
Consewation considerations
SUMMARY
LITERATURE CITED
APPENDICES List of Tables
Table 1 Distribution of West Indian mb-dwelling vireos (Vireo spp.)...... Page 6
Table 2. Collection locales, date, sample size, recordist (and institution) and location
abbreviations (used elsewhere in the study) of Vireo crassirosrris reçordings...p age 15
Table 3. Primer sequences used in the study of Vireo crussirosfrisand congeneric species
...... Page 26
Table 4. Nurnber of syllable types (rnemes of length 1) recorded and the corresponding
number of unique (private) syllables in each Yireo crassirosrris population ... .page 33
Table 5. Syllable measurements: minimum and maximum fiequency, maximum syllable
length, percentage hi& fiequency (> 8000 Hz.) and long syllables (> 0.3 seconds)
in eight populations of Vireo crassirostris...... Page 35
Table 6. Number of syllables (N = 80) and percentage of syllables shared between
populations of Vireo crassirostris...... a 3 7
Table 7. Mme identity and diversity within Vireo cvassiroslris populations. (Merne identity
(I), unbiased estimate of meme identity (1), effective number of rnemes (se) and
unbiased estimate of effective memes (sJ are shown)...... page 42 Table 8. Merne flow vatues (Na)for seven populations of Vireo crassirostris populations
...... page 43
Table 9. Populations of Vireo crassirostris sarnpled in the analysis of number of song types per
population and number of song types shedby ai least two individuals ...... page 45
Table 10. Haplotype designation and variable nucleotide positions (L-sûand) in 17 Vireo
crassirostris mtDN A control region sequence haplotypes fkom 8 populations......
...... pa 53
Table 11. Percentage fiequency of haplotypes identified in eight populations of Vzreo
crassirostris...... a 5 4
Table 12. Haplotype diversity (h) and nucleotide diversity (n)within eight populations of
Vireo crassirosrris ...... PWe 59
Table 13. Estimates of gene flow (N,) in populations of Yireo crassiroslris in the northern
Bahamas, Turks and Caicos Islands and for the Bahamian archipelago...... page 60
xii List of Figures
Figure 1. Distribution of the Thick-bUed Vireo (Vireo crussirostris) in Bahamas, Turks and
Caicos Islands, Caylnan Islands, Isle de la Tortue off north-west coast of Hispaniola,
and Paredon Grande Cay off north-east Coast of Cuba ...... page 8
Figure 2. Map of the Cayrnan Islands. Vireo crassirosrris is found on Grand Cayrnan
and Cayman Brac but has been extirpated on Little Cayrnan ...... Page 9
Figure 3. Schematic representation of rnitochondrid genes in the vicinity of the control region
and locations of prirners used in the study ...... Page 27
Figure 4. The longest syllable (.6062 sec) as recorded for a Vireo crassirostriu, Andros Island,
Bahamas...... Page 34
Figure 5. A neighbour-joining tree based on Jaccard's coefficient showing level of syllable
sharing arnong populations of the Thick-billed Vireo (Vireo crassirosfris)with
Vireo crassirostris approxirnans (Ida Providencia, Colom bia) as outgroup......
...... Page 38
Figure 6. UPGMA phenogram based on clustering of Jaccard's coefficient showing level of
sy llable sharing arnong populations of Vireo crassirosfriswith V. c. approximans
(Ma Providencia ,CoIombia) as outgroup...... Page 39
xiii Figure 7. Maximum parsimony tree showing level of syliable sharing among populations of the
Thick-billed Vireo (Vireo crassirosfris)with K c. appmximans (isla Providencia,
Colombia) as outgroup. Heuristic search, branch and bomd and exhaustive methods
dl produceci the same tree...... Page 40
Figure 8. SimiIarity in Song types among Vireo crassirosfrispopulation ...... page 46
Figure 9. Temporal sharing of song types by chronologicd populations of Yireo crarsirostris
recorded on Grand Cayman in 1971 and 1997 ...... Page 48
Figure 10. A mix of song types within a singing bout by a Vireo crassirosfri.srewrded on New
Providence ...... page 49
Figure 11. Chronological development of song types in a Vireo crassiroslris recorded on
Providenciales (1 8 June, 1997) demonstrating one way that Song types are changed
after one Song type has been uttered 25 to 50 &es ...... page 51
Figure 12. Minimum spanning tree showing the relationship of haplotypes in eight populations of
Vireo crassirostris...... Page 55
Figure 13. Neighbour-joining tree showing genealogicd relationships among control region
haplotypes in eight populations of the Thick-billed Vireo (Vireo crassirostris)with
the White-eyed Vireo (K griseus) used as the outgroup taon...... pages 56 -57
xiv Figure 14. UPGMA phenogram with Kimura two-parameter correction of control region
sequences of Vireo crassirosZris and congeneric Weos: White-eyed Virw (K
griseus), Mangrove Vireo (K pallens) and Flat-biiled Vireo (K nanus)....
...... f...... f...... Page 62 List of Appendices
Appendix 1 Example cdcdation of merne identity for yireo crussirostris
using the population fiom Abaco Island. Bahamas ...... Page 96
Appendix 2 Specimens of Vireo crassirosfris and other Weos used in the study...... page 97
Appendix 3 Ykeo crassiro.sfrissequences (L-strand) ...... Page 99
Appendix 4 Variable sites .Vireo crossirosfris sequences (L-stmnd) ...... page 106
Appendix 5 Nucleotide composition of Vireo crassiro.strk...... page 107
Appendix 6 Hurricane data for West Indies with emphasis on the range of Vireo
crausirostris...... p 1 08 Introduction
"Population studies offer a researcher the opportunity to acquire a deep and familiar understanding of a
species. The present study of cultural and genetic evolution in the Thick-billed Vireo has given me the
chance to infer its evolutionary story, albeit a relatively short one in evolutionaxy time. Now is the tirne
to share that story." (Marlene Walker 1998)
INTRODUCTION
In this population sîudy of the Thick-billed Vireo (Vireo crassirostris), 1 examine two evolutionay pathways, cultural and genetic, to detect patterns of divergence among populations of this species.
Islands, being isolateci in time and space, are naturd laboratories for the study of evolution while sedentary taxa with allopatric distributions in island environrnents (Grant and Grant 1996) are ideal candidates for the study of evolutionary processes. The Thick-billed Vireo, an island resident with the largest number of insular populations in the subgenus Vireo in the West Tndies meets the above criteria.
In this thesis, the effects of geographic isolation on genetic characters and bioacoustic elernents of Vireo crassirostris are assessed using the mitochondrial control region (Domain 1) and song elements, respectively. The direction, severity and fiequency of htmicanes in the West lndies are andyzed with the view to understanding their potential impact in dispersion of Thick-biiled Vireos throughout their geographic range. introduction 2
Cultural and genetic evolution
Cultural evolution, as it pertains to birdsong involves the transmission of units of song, or mernes, a term coined by Richard Dawkins (1 976) for any cultural trait capable of being transmitted. Mundinger
(1980) first app1ied the tenn to birdsong in terms of individual syllables, while Lynch et al. (1989) broadened the definition to include not only an individual syllable (merne length l), but also a group of comected syllables (memes of vmng lengths) and an entire Song (song meme). The meme in the population memetics model is analogous to the gene in population genetics -ch and Baker 1993).
Consistent with neutral theory the genetic mechanisms of migration, mutation, &fi and seiection have their memetic counierparts in cultural evolution (Cavalli-Sforza and Feldman 1981, Lynch and Baker
1993).
Mutation in cultural evolution proceeds more quickly than genetic mutations (!nce et al. 1980, Jenkins
1977, Lynch 1996) through the relative ease that singers can miscopy syllables, and dso through the generation of new syllables. While the merne pool itself is finite at any given tirne, the mechanisms of memetic drift and migration (Lemon 1975) dong with cultural mutation affect changes in the merne pool available to the population. Simultaneously, but more rapidly, the Song type pooI is changing due to both point mutations (changes in the basic elements), and recombinations, which are the arrangement of the basic elements (Lynch 1996). Marler and Peters (1 982) refer to these mutations as elernental and combinatorid improvisations, respectively. Because the possibilities are endless for both types of mutations, cultural evolution fits the infinite alleles model applied to genetic evolution, which States that any new mutation is a new mutation not previously seen in the population (Nad and Clark 1989). The mode1 assumes that the population is finite, and that both drift and mutation occur. tntroduction 3
Speed of cultural change is species-specific and depends on population size, duration of cultural generations, mortality rates, accuracy in copying (Lemon 1975, Ince et al. 1980) and the number of models singing the same song type in proximity to young birds. In con- to genetic evolution which proceeds vertically fiom parent to offsprin& cultural transmission can be acquired vertically, horizontally (fiom other adults of the same generation) and obtusely (crossing generations) (Cavalli-
Sfona and Feldman 198 1).
Published bioacoustic and genetic data are lacking for the Thick-billed Vireo, although data are available on distribution (Kirkconnell and Garrido 1991, Bond 1985, 1978) and phenotypic geographic variation
(Buden 1985). To my knowledge, there are few studies at al1 investigating molecular and Song data concornitantly. Exceptions are cytochrome b sequence data with analysis of vocalizations in the
Paiaearctic chiffchafTcornplex (Phylloscopus collybira) (Helbig et al. 1996) and DNA-DNA hybridizaîion and vocalizations in herons (Ardeidae) (McCracken and Sheldon 1998). Gariido et al.
(1997) compare taxa in the endemic West indian Stripe-headed Tanager (Spindalis spp.), using vocalizaîions and morphological data, while within the same genus, Klein (unpubl.) infers phylogenetic relationships using plumage patterns and mitochondria1 DNA (mtDNA) sequences. Lynch (1 996) has suggested that comparative studies between genetic markers and song elements wodd be instructive if the hypervariable (Vigilant et al. 1989) area of the control region (mtDNA) is studied in conjunction with
Song analysis.
The rniiochondrial DNA molecule is fiequently used to assess population stnicturing due to its hi@ mutation rate (Quinn and Wilson 1993, Baker and Marshail 1997). The non-coding control region, which Introduction 4
evolves three to five ximes more rapidly than oîher areas of the mtDNA genome (Aquadro and Greenberg
1983) has been selected to facilitate analysis for a relatively recent colonization event, inferred fiom
biogeographical material (Pregill and Olson 1981). Avian population studies ushg control region include
work on Dunlin (Calidris alpina) (Wenink et aI. 1994, Wenink et al. 1 996), Ruddy Turnstone (Arenaria
intetpres) (Wenink et ai. 1994), guillemots (Cepphus spp.) (Kidd and Friesen 1998) and finches
(Carduelis chloris) (Marshall and Baker 1997, Merila et al. 1997) and Fringillu spp. (Marshall and Baker
1997).
Profile of the Thick-billed Vireo
The Thick-billed Vireo is a non-migratory, primarily insectivorous, scrub-dwellhg species in the
subgenus Vireo (Vireonidae). Although divergence tirnes are not known, the fmily is thought to be of
ancient origin (Avise et al. 1982). Typical of the family, al1 mernbers have IO primaries, although the
tenth may be vestigial.
The thick-bill is a small(13 g) dull-plumaged bird with an olive-green back and wings, two whitish wing bars; whitish edges to the wing feathers; a slightIy hooked and thick bill relative to the White-eyed Vireo
(Yireo griseus); bright yellow through the forehead and supercilium and a dark line through the lore giving the appearance of "spectacles"; wiîh underparts entirety yellow to yellowish; and the undertail coverts bright yellow (P. E. Bradley 1995). The immature bird has olive upperparts and olive yellow underparts; the dark line in the lore is absent (P. E. Bradley 1995). In the Cayman Islands it favours open woodland, low scrub vegetation, bushland and Buttonwood Mangrove (Conocarpus erecfus)
(P. E. Bradley 1995) and in the Bahamas occurs in scrubland and xeric to mesic woodlands (Buden
1985). Introduction 5
The Thick-bikd Vireo has a heterosyuabic Song which consists of several different syllables utiered
sequentidly, in contrast to monosyllabic song, in which songs include fi-orn 1 to 20 syilables, al1 of
which are alike in a given Song type, as in Hutton's Vueo, Vireo hutroni (Baril, in Litt.). On the other
hand, chatter song, a terrn coined by Barlow (unpubl.), is usually comprised of one syllable per Song but
repeated a variable nurnber of times in rapid succession, as demonstrateci by Vireo crassirosrris
upproximans. The signature sound of the thick-bill is a non-musical buzy, buny syllable type which it
incorporates into the rnajority of its songs. This syllable type is fiequency modulated and Sung at varying
frequency and duration. In the field, within that part of the West indies where it occurs, songs
distinguished by the buzzy syllable are clearly recognizable as a thick-bill, unless it is winter when the
Song of the Thick-billed Vireo could only be coiifised with that of the migratory mainland, White-eyed
Vireo ( Vireo griseus).
Distribution and current raxonomy
The Thick-billed Vireo has the most fiagmented distribution of island endemics in the subgenus Vireo.
Among the eight smb-dwelfing sedentary taxa in the sub-genus that fil1 the same ecological niche throughout the West Indies, it is the only species that occurs on more than one island (Table 1) with its distribution separated by the Cuban land mas. In contradistinction to the rnajority of these taxa which are highly divergeci fiom the ancestral 'white-eye' type, the Thick-billed Vireo vocally (Hopp et al. 1995,
R. A. Bradley 198 l), phylogenetically and phenotypidly resembles the White-eyed Vireo (K griseus). Introduction 6
Table 1. Distribution of West Indian scrub-dwelling weos (Vireo spp.).
Species Island Status Thick-billed Vireo Bahamas; Caicos 1 Y: crassirostris Islands, Turks and 1 Caicos; Cayman I Islands; Paredon I Grande Cay, Cuba; Isle I de la Tortue (Haiti) 1 Jamaican White-eyed Vireo K modeslus Blue Mountain Vireo K osburni Cuban Vireo K gundlachii Flat-billed Vireo IHispaniola St. Andrew Vireo St. Andirew (San
Cozumel Vireo Cozumel Island Vireo bairdi Puerto Ricm Vireo Puerto Rico V. latirneri I Introduction 7
The thick-bill occurs widely (Fig. 1) throughout the Bahamian archipelago, and also in the Cayman
Islands (V-c. aiieni [Co~y])on Cayman and Cayman Brac (Fig. 2). Cayman birds have been
placed in the nominate fom Vireo crassirosfris crassirosfris by Bryant (1859), and in K c.flavescens by
Ridgeway (1 887). Cory (1892) consideredflavescens to be a synonym of aileni giving its range as the
southem Bahamas and the Cayman Islands. Buden (1985) retains the name alleni for the Cayman
population, based on morphologicai variation. Although presurned extant when Johnston et ai. (1971)
listed the avifauna of the Cayman Islands, it has been extirpated on Little Cayman (Diarnond 1980,
P. E. Bradley 1995).
Within the 714 islands and cays comprising the Bahamas, the only two recognized sub-species of mck- billed Vireo are the nominate forni, K c. crassirosfriv found north of the Caicos Islands and
K c. stalugmium, described by Buden (1 985) for the populations occurring in the Caicos Islands in the
Turks and Caicos (the species is not found in the Turks). Buden (1 985) notes colour differences in the
Bahamian populations with lighter yellow birds p~imarilyon northern islands (north of the Crooked
Island trench, 23O N and 74"W) and darker yellow birds to the south of this trench, suggesting the effects of isolation. The thick-bill generally occws on small islands, the largest being Andros (excluding the rest of the Andros archipelago), which is approximately 7500 km2and considerably larger than al1 other islands on which the thick-bill is found. Fifieen islands in the Bahamas have areas greater than 65 km2, whiIe the rest are smaller, including thousands of tiny cays (Miller 1978). The species is found on rnost of the islands, even on cays as small as Allan's Cay (91 -44 m by 152.4 m) (Anthony White, in Litt.). introduction 8
w a- \ Caribbean Sea 3
Fig. 1. Distribution of the Thick-billed Vüeo (Y crassirostris)in Bahamas, Turks and Caicos Islands
(only found in Caicos Islands), Cayman Islands (Orand Cayman and Cayman Brac), Isle de la Tortue off the north-west coast of Hispaniola, and Paredon Grande Cay off the north-east coast of Cuba. Note that the species is confined to islands of the NE West Indies and that Cuba separates the Cayman Içland populations fiom the otha populations. The Crooked Island, Mayaguwa and Caicos passages are marked by deep sea trenches. The Bahamas include 7 14 islands and cays (dl not shown). Ida Providencia (not shown) is in the south-western Caribbean, 670 km due south of Grand Cayman Island. Populations fiom islands included in this study are labeled. Providenciales and Middle Caicos (Turks and Caicos Islands) are # 1 and #2, respectively. Grand Cayman and Cayman Brac (Cayman Islands) are #3 and #4, respectively. Adapted from Map of the Gulf, Caribbean and Atlantic Coast Area, Harnmond Inc.,
Maplewood, New Jersey. Introduction 9
Little Cayman / &@CaymanBrac
Grand Cayman Caribbean Sea
Fig. 2. Map of the Cayrnan Islands. Kirno crussirosrris is found on Grand Cayman and Cayman Brac but has been extirpated on Litrie Cayman. Grand Cayman is 1 17 km west-southwest of Little Cayman, which is 7.5 km due west of Cayman Brac. Introduction 10
WMe presumably absent fiom the large Greater Antillean islands proper, a breeding population of 50-60
thick-bills was confirmed in 1997 on Paredon Grande Cay off the north-east coast of Cuba (Arturo
Kirkconneii and Orlando Gamido, in Litt.). The species is aIso found on Isle de la Tortue (K c. tortugae)
off the north-west coast of Haiti (Hispaniola). Wetmore and Swales (193 1) did not find it surprishg that a
'crassirostris' resides on Tortue' since the distance between Tortue and Great inagua (Bahamas) is only
80.47 km; however, they found it remarkable that the bird had not been found on 'mainland' Haiti, only
8.05 km away.
Thick-bills have also been reported as vagrants (non-breeding) on the south-eastem coast of Flonda and the Fionda Keys (Smith et aI. 1990, Langridge 1988, Abramson 1974). Since 1992, &ter Hunicane
Andrew leveled Bill Baggs Cape Florida State Recreation Axa, reports in Florida have decreased (P. W.
(Bill) Smith, in Litt.), for it was the primary location where thick-bills and birders intersected.
In addition, Kc. uppximans, living on Providencia Island (Colombia) (not show) in the south-western
Caribbean was placed in the 'crassirostris' complex by Bond (1965). This 'outlier' crassiros~rissings only chatter song, contra the heterosyllabic Song of other 'crassirostris' (Barlow 1995)' and is largely indistinguishable fiorn that of the Mangrove Vireo (Vire0 pallens) of the Caribbean coast of Central
America and Mexico to which it is cleady closely allied (Barlow, unpubl.).
A biogeographical perspective of the West Indies
In this section I set the stage for interpreting genetic and vocal diversity among eight populations of thick- bills fiom a biogeographical and geologicd perspective. in particula. 1 am interesteci in the degree of Introduction 1 1
isolation, both in time and space, of the islands on which this species lives. This necessarily involves the emergence of various land masses, and previous land connections that may have facilitated range expansion. A cornpreiiensive study of the geology of the Caribbean region is not within the scope of this work. Ancient geology is not relevant to the distribution of the species as its evolutionary history post- dates the older geological history of the region.
1 now clari& what is meant by a nurnber of terms used in the context of this study. The 'Bahamas7 refer to the islands and banks of the Bahamas, formerly a British Crown Colony that achieved its independence in 1973 (Buden 1987). The term Bahamian archipelago refers to the Bahamas and the Turks and Caicos
Islands. The Turks and Caicos Islands are located at the southern end of the Bahamian chah of islands and are an independent political entity fiom the Bahamas (Buden 1987) and a British Crown Colony. The northern Bahamas refer to those islands of the Bahamas north of the Crooked Island Passage, and include the islands of Andros, Abaco, New Providence and San Salvador (referred to in the present study). The southem Bahamas include al1 islands south of the Crooked island Passage, including the Turks and Caicos
Islands.
Changes in sea-level during the Pleistocene resulted in varying island size in the Bahamian archipelago over time (Faaborg 1985). The Bahamas wzre totally submerged during the last intaglacial, for which estimates range fiom 65,000 - 80,000 ybp (Pregill and Olson 1981) to 120,000 ybp (Morgan 1989). This suggests that thick-bills were living elsewhere, on higher ground, or that their divergence is of more recent origin. Schuchert (1935) noted that birds are less migratory in warmer climates and therefore in contrast to cold climates may be a good index of former land connections. Coalescence and separation of land masses have cIearly resulted in different biogeographical histones for the islands of the archipelago with Introduction 12
changes in sea level significantly influencing the historical dispersal of the avifauna of the Bahamas. The
Bahama Banks at Iow sea level 17,000 years ago, increased the total Bahamian land mass by an order of
magnitude greater than the area of al1 existing islands tociay (Pregilf and Olson 1981) with these
Bahamian islands acting as stepping Stones between Florida and the Greater Antillean islands of Cuba and
Hispaniola (Faaborg 1985). Geologists believe that southeastern Cuba and northem Hispaniola were
connected in the Eocene but separated later in the Tertiary (Hedges 1996).
GedogicaIly the Bahamas are divided into northem and southem regions, each with its own distinctive
history (Schuchert 1935). The older, continental islands of the northern Bahamas are part of the Florida-
Bahamas Platform of the Norîh American Plate (which underlies the Turks and Caicos) and once were
connected to the Cuban foreland. The younger, oceanic islands south of the Crooked Island Passage are
of volcanic ongin. Crooked Island Passage (2,743 rnetres deep) Iies between Long Island and Crooked
Island, and Mayaguana Passage (1 800 - 4860 m) lies between the Crooked - Acklins Bank and
Mayaguana to the south (Buden 1987, Schuchert 1935). The presence of these trenches is evidence that
the islands which they separate have never been connected in times of lower sea levels. P rovidenciaies,
Middle Caicos and the other Caicos islands are separated fiom the rest of the southeni Bahamas by the
Caicos Passage (2592 - 4095 m) which lies between Mayaguana in the north and the Caicos Islands to the south. The Caicos Islands have clearly been connected to each other in recent times when sea level was lower. Flying over these islands, it is difficult to distinguish their shorelines and a large land mass spreacling in an arc is evident. in contrast to the northem Bahamas, where coalescence encouraged the miwig of avifauna, the deep trenches among the southern Bahamas preclude vicariance. Most likely islands separateci by the deep trenches had less mixing across populations unless, of course, in times past, sedentary birds were more mobile and flew across the narrow gaps. Introduction 13
Thick-bills inhabiting the Cayrnan islands are geographically farthest removed fiom Bahamian
archipelago thick-bills and hence least iikely to receive dispershg thick-bills fiorn the archipelago
populations. The Cayrnan chah derives fiom the prehistonc geological submarine formation known as
the Cayman Ridge, which formerly extended fiom Central Arnerica to Cuba (Jones 1994, Johnston et al.
197 1, Johnston 1975). The Caymans rose above sea-level in the middle Miocene, ca 30,000,000 years
ago and were part of the Sierra Maestra mountain range of Cuba (Schuchert 1935). Any movement among these islands must be by over-water dispersal, as the islands of this part of the West Indies have never coalesced.
Objecf ives
The primary objective of tiiis study is to defme patterns of divergence in cultural and genetic evolution in populations of the Thick-billed Vireo. The hypothesis tested is that sedentary populations are divergent both cdturally and genetically due to their geographic isolation. The following questions are addressed:
What are the effects of geographic isolation on genetic characters and bioacoustic elernents in
populations of the Thick-billed Vireo?
Are the findings consistent with currently accepted taxonomy?
Are the fmdings of song type variability consistent with the 'loss of contrast' hypothesis? Materials and Methods: Bioacoustic AnaIysis
BIOACOUSTIC ANALYSIS
Field equipment and merhod of recording
Severai investigators have made field recordings of Thick-billed Vireo song. Recordings of vocalizations (mostly song) used in the present study are atbibuted to Jon C. Barlow (JCB) of the Royal
Ontario Museum (ROM)who taped thick-bills on Andros (1 97 1) and New Providence (1 971, 1974) in the Bahamas and Dr. Steve Hopp of the University of Arizona on Abaco (1993). JCB and I recorded
Song on New Providence (August, 1996) and the Cayrnan Islands (May 1997). Recordings were made by Mark Peck (MP) and myself (MRW) on Abaco, Andros, and San Salvador (August 1996), and
Providenciales and Middle Caicos, Turks and Caicos (June 1997). For details on collection locales, recordists and sample sizes see Table 2.
In 1997 recordings were made with a Sony Pro Walkman D6C using a Sony Electret Condenser Stereo
Microphone and a 33 cm Sony Reflector PBR-330acrylic parabola. The 1996 Song data was recorded with a Sony Pro Waîkman or a Marantz PMD 220 Professional Cassette Recorder with condenser microphone (2 speedl3 head system) and windscreen. Tapes used were usually Sony high quality chrorniurn dioxide. Recordings of vocalizations were annotated with date, time, location, individuai bird nuinber and narne of the recordist. Songs fiom the early to mid 70's were recorded on a Uher 4000
Report L reel- to-reel recorder at 19 cm per second with Audio-Technica 128 microphone and 46 cm
Dan Gibson acrylic parabola.
When possible, recordings were of spontaneous song, however bief playback of previously recorded
Song of other birds on an island, or from a singer's own repertoire just recorded, were used to induce Materials and Methods: Bioacoustic Analysis 15
Table 2. Collection locales, date, sample sk, recordist (and institution) and location abbreviation (use4 elsewhere in the study) of Vireo crassirostris recordings. Vïreo crassirostris approximans (Providencia Island, Colombia) is the outgroup for the bioacoustic analysis. Recordists' names are abbreviateda.
1 Population 1 Dates and Number of Birds Recorded Recordist and institution 1 Abb. 1
New Providence 9 - August 1996 JCB and MRW (ROM) NP 4 - July 1971 JCB (ROM) 2 April; 1 May; 2 - October 1966 - - Cornell Laboratory of 5 - May; 1 - June 1971 Ornithology
Andros Island 14 - August 1996 JCB and MRW (ROM) 4 - July 1971 JCB (ROM)
Abaco Island 8 - August 1996 MRW and MP (ROM) 9 -0ctober 1993 Steve Hopp (U. of Arizona) 1 San Salvador 1 18 -Aupst 1996 MRW and MP (ROM)
Grand Cayman 6 - May 1997 JCBandMRW(R0M) GC
5 - Apnl 1971 JCB (ROM) 9 - April 1974 JCB (ROM) 1 Cayrnan Brac JCB and MRW (ROM) ICB I Paredon Grande Cay (Cuba) ? - Dec. 1996, Arturo Kirkconnell I PGC l ?-Malch, 6-May 1997 (Museo Nacional de Historia Natural, Cuba Providenciales. Turks and Caicos 1 7 - June 1997 Middle Caicos, Turks and Caicos 2 - June 1997 MRW and MP (ROM) Providenciales, Turks and Caicos _ J,, 1996 IC I Bruce Hallett (recordist), provided by Anthony White Providencia Island (Coiombia) - May 986 JCB (ROM) Vireo crassirostris approxirnans a: Jon C. Barlow - JCB; Mark Peck - MP; Marlene R. Walker - MRW; 3M - Royal Ontario Museum Materials and Methods: Bioacoustic Analysis 16
shging when season and/or theof day was not conducive to spontaaeous song. A second tape recorder and speaker was used for playback.
Recording parame fers
The acoustic environment ( how much singing, frequency, insect interference, number of species singing), weather conditions (wind, rain, cloud cover), season (breeding, post breeding, winter) and the of day dl affect recording conditions. in addition, technical problems can trouble an investigator. Of course human error and the individual expertise of the recordist may play a role, There may be recording aberrations as a result of wind, direction of the bird, and microphone direction. The amount of recording per bird varies with tirne of day, breeding activities, whetlier the bird stays in the same location and continues to sing and the arnount of time the researcher spends on recording the individuai. Therefore 1 have lengthy recordings for some individuals and minimal amounts for others. Depending on population density and accessibility, some populations are easier to record, so that the number of individuals recorded per population also varies. Because there is a high degree of variation both within a population and in an individual's repertoire, large samples are necessary and long recording periods are needed to capture their Ml repertoire (Marler 1960).
Definitions
Syllables (memes of length 1, elements, notes) are defined as the smallest temporal unit of a song, and
Song consists of a group of syllables separateci eorn another group by a pause longer than the pauses between the notes (ThieIcke 1969). A syllable type is an individual syllable and al1 of its variants. A Materials and Methods: Bioacoustic Analysis 17
Song type is defined as a repeated mangement of syllables that cm be recognized as a unique pattern.
In addition, each Song type may have 'variations on a theme', which mauitain the integrity of the Song type while displaying some difference fiom the 'master' Song type.
Sound Iaboraiory analysis
Audiospectrographic assay of songs of 130+ males was done to assess level of similarity among syllables of eight geographically isolated populations of the Thick-billed Vireo (Cayrnan Islands: Grand
Cayman, Cayman Brac; Bahamas: Abaco, Andros, New Providence, San Salvador; Cuba: Paredon
Grande Cay; Turks and Caicos: Middle Caicos and Providenciales are treated as one population).
Recordings were analyzed in real time using the wide-band setting of a Kay Elemetrics DSP 5500
Sound Station in the Sound Laboratory of the Centre for Biodiversity and Conservation Biology, Royal
Ontario Museum, Toronto, Canada The time axis was set to 2 sec. and fiequency intervals were fiom O
Hz to 8000 Hz. The 16 Khz setting was used when syllables were equai to higher than 8 Khz in pitch.
Vïreo crassirostris approximans of Providencia Island (Colombia), a chatter singer and probably not a crassirosiris (Barlow, in Litt.) was used as the outgroup taxon for the Song assay (N = 1 1, recorded by
JCB [1986]). Songs were evaluated according to: number of syllables per Song, syllable duration, Song length, intersyllabic interval, fiequency range (pitch) and change in fiequency. Each sonagram printed was compileci with its andysis, dong with its bird identification nwnber, location, number of repetitions, the date, tape number and recorder counter number. Each syllable was colour-coded as per population, noting the bird number, fiequency range, syllable duration, and tape and counter number.
Syllables not readily assigrtable (faint, bIurry) to a category because of recording problems were not included in the analysis. Syliables which were visible at the high end of the 8 Khscreen were re- examined on the 16 Khz screen. By visual cornparison (gestalt) (Lang and Barlow 1987, Lynch and Materials and Methods: Bioacoustic AnaIysis 18
Baker 1994) each syiiable was examuied and compared to determine if it was shared by other birds in the same population and by birds in other populations, and then charted on a syllable/population matrix.
1 assumed sirnilarity unless the syliables were cleady different as apparent minor variations may be due to the recording constraints as noted above. To provide fiirther verification of the wide band comparkons, narrow-band syllables were also used, dong with duration and fkquency measurements.
Traditionally most investigators rely on the wide band setting in their classification of syllables.
Distinguishing different syllables and çomparing them with presurned homologies in other populations may be subjective (Hauser 1996). Many syllables are similar in shape but differ in fiequency and length and accordingly represent a continuum of variants. In addition, implications of the "uncertainty p~ciple"(Beecher 1988) suggest that accurate spectrographie measurernent is constrained by fiequency and duration as bot. dimensions Vary sirnultaneously. With this in minci, an investigator must make a decision as to when to 'Iurnp' syllables and when to treat them as different fiom each other. Excessive lumping overemphasizes sharing, however, recognizing too many distinctive syllables reduces sharing.
Therefore, balance between lumping and differentiating is required. When taking recording considerations into account, some syllables appear different on a sonagram but in reality would be the same if recording conditions were always equal.
Chsfer anaiysis
The level of sharing was caiculated using Jaccard's Coefficient (SJ (Sneath and Sokai 1973).
Jaccard's is a binary differentiation between the presence (denoted 1) and the absence (denoted 0) of a syllable. Only positive matches are used in the computations of sharing, since negative matches may Materids and Methods: Bioacoustic Analysis 19
falsely indicate that the syiiable is not present in the population whereas it rnay be present but not recorded. Jaccard's Coefficient (S,) is defined as
where nx is the number of matches between J and K, and the nwnber of mismatches is u. This andysis produced a distance matrix. The resultant values were clustered (Lang and Barlow 1987) using UPGMA
(ünweighted pair group method with arithmetic mean) to construct a phenogrm (distance tree) using
SIMQUAL (similady for qualitative data) and SAHN (sequential, agglomerative, hierarchicai and nested) on the NTSYS (1992) software program to determine the magnitude of divergence among
'crassiros~ris'populations.
Neighbor-joining (Saitou and Nei 1987) using a transformation (negative log) on NTSYS (1992) was performed to consbuct a neighbour-joining tree. Coplienetic values were anaIyzed using a matrix cornparison with 1O00 permutations. The cophenetic correlation coefficient indicates the level of confidence one can expect in the transformed data fiom the original simiIarity matrix (Sneath and Sokal
1973).
Maximum parsimony analysis
Maximum parsimony using PAUP (Swofford 1993) was performed on the data set using the heuristic search, branch and bound, and exhaustive methods. Bootstrapping of 1000 replicates was done, and both the consistency (CI) and retention (RI) indices were calculated. Materials and Methods: Bioacoustic Analysis 20
Sta f is f ical anabsis
Meme diversity, or the effective number of rnemes (sJ(Lynch and Baker 1993) within populations was
estimated for al1 populations, except for the Paredon Grande Cay sarnple of birds which çould not be
identified individually in the recordings. Neither was meme diversity calculateci for the 'approximans'
population which does not have a typical 'crassiros~ris'song. An initial calculation of meme identity (1)
is required. Individual occurrences (how many birds utter each syllable) per population is required for
these masures. The fiequency of merne occurrences per population is obtained by 1) counting the
number of times each meme (syllable) is uttered, 2) adding up ail occurrences, 3) dividing die total
number of occurrences per merne by the total number of memes, 4) squaring each calculation fiom
number (3); and 5) adding up dl squares equals meme identity (See Appendix 1).
The concept of meme diversity folIows fiom the infinite alleles mode1 which assumes that any new
meme arising in a population is a new one not previously represented in that population (Kimura and
Crow 1964). Merne identity gives the probability of randomly selecting two memes which are the same
and is defined (Lynch 1996) as
where S is the number of different meines in a population of size N and the fiequency of the ph merne is pk ('F2above is p2). This statistic is analogous to homozygosity or gene identity in population genetics
(Nei 1973). To correct for sample size, an unbiased estimate of 1 can be obtained ( f) Materials and Methods: Bioacoustic Analysis 21
where n is the sarnple size, s is the number of memes in the sarnple, and xk is the fiequency of the ph
meme in the sample (Nei and Roychoudhury 1974, Gregorius 1987). Using the above striristics fiom
meme identity, rneme diversity can be calculated as
where s, is the effective number of memes. This is a better indicator of meme diversity than the actual nurnber of memes because effective number of mernes is independent of sarnple size, whereas actual number depends on sarnple size (Lynch 1996). An unbiased estimate of meme diversity is therefore
Meme flow (N,) or migration was calculated using the number of unique (private) sytlables per population according to Slaîkin (1985)
wherei(1) is the average fiquency of syllables unique to a population, and a = -0.505 and b = -2.440 are empirically derived constants (see Lynch 1996). Estimates of meme flow were adjusted by the ratio of25 (SI& 1985). Materials and Methods: Bioacoustic Analysis 22
Merging of data sers
Songs of Thick-billed Vireos recorded in the 1970's were combined with recordings fiom the 1990's because upon cornparison 1 found that most syilables were represented in both data sets (87% sharing for New Providence, 94.87% Andros). Barlow (April 1971, August 1974) recorded thick-bills on Grand
Cayman and obtained a good representation of syllables. In contrast between May 16 - 20 1997, thick- bills on Grand Cayman sang little and did not respond to playback, while on Cayman Brac (144.84 km
NNE of Grand Cayman), they were in full Song suggesting different timing of the breeding cycle, perhaps due to lengthy drought conditions (P. E. Bradley, in Litt.). On Grand Cayman where incubation was underway, Song was greatly reduced. Given the relatively high level of sharing between the 1970's and 1997 (68.42%), I merged the two data sets for Grand Cayman.
Song Qpe analysis
Following the syllable analysis, each population was re-examined to determine the number of song types recorded and the incidence of each Song type. Cornparisons of within and between population data were undertaken. This analysis of song in Vireo crussirostris examines Song types in relation to the 'loss of contrast' hypothesis (Thielcke 1973).
Differentiating songs into Song types poses problems similar to those encountered with the differentiation of syllables as discussed above. Because each Song consists of fiom three to fourteen Materials and Methods: Bioacoustic Analysis 23
syllables this process is exacerbateci. If cornparhg similarity in song types is difficdt within one temporal period within one population, it is rnuch more difficult to compare the same population between time periods (Ince et al. 1980). Deciding whether a Song type is shared within populations, at the same time or across time is a subjective decision by the investigator. 1 have made my decisions based on maintenance of the integity of the 'master' Song type which means that the rnajority of syllables are present at a similar fiequency and duration, and in the same arrangement. Any major syllable or element must be present. The visual Gestalt when comparing songs should suggest 'sameness'. Due to the high degree of variability in Song types both within and between populations, and the relatively srnall sample sizes, I have not undertaken phenetic distance analysis for Song types. Materials and Methods: Genetic Anaiysis
GENETIC ANALYSIS
Thick-billed Vireos were netted (2.7 m or 3.6 m mist nets) and 0.25 ml of blood taken fiotn the brachial vein of each specimen in a sy-ringe containing EDTA and preserved in individual vials containing 95% ethanol. Birds were re1eased in a procedure that lasted between two to five minutes. Each sample was numbered and labeled, with records kept of the date, location, sex and age of each bird. These records are entered into the data base of the Centre for Biodiversity and Conservation Biology of the Royal
Ontario Museum (Appendix 2).
Total DNA was isolated fiom collecteci blood sarnples using standard proteinase K-phenol-chloroform methods (Sambrook et al. 1989). Following two extractions with buffer-saturated phenol and once with chlorofom: isoamyl dcohol (24: l), half of the sarnples were precipitated with sodium chloride and
95% ethanol, and resuspended in distilled water.
The amplification reaction protocol, according to the manufacturer's insbuctions (Perkin-Elrner
Amplicycle TM) contained 18.3 pl distilled H20;2.5 pl buffer; 1.0 d NTP; 1.O pl each primer; 0.2 pl
Taq DNA polymerase (enzyme); and 1.0 pI DNA template for each sample in a 25 pl volume. Two enzymes and two buffers were used in independent amplifications; Perkin-Elmer Amplitaq or Materials and Methods: Genetic Analysis 25
Boehringer-Mannheim Taq DNA polymerase, and Erica Hagelberg (10 x EH) or Boehringer-Mannheim bufférs, respectively. Versatile primers outside the control region were used in a polymerase chah reaction (PCR). Initial denaturing at 95OC for 2 minutes was followed by amplification through a thermal cycle of 94OC for 45 s, 45OC for 45 s, and 72°C for 90 s, repeated 35 times. The primer 16500
(Quinn 1992) (Table 3) was used in combination with ADL (Vicki Friesen, unpubl.) or LPRO frara
Paton, unpubl.) (Table 3). Counter-intuitively, universal primers fiom outside the control region for amplification produced better sequence results than vireo-specific primers. (For location of genes in the mitochondrial avian genome refer to Fig. 3).
Arnplified products were isolated by electrophoresis through a 2% agarose gel (0.8 g agarose dissolved in 40 ml TA buffer; heated one minute) with ethidium bromide (2.0 pl) added for the purpose of visualization under ultra-violet Iight. All electrophoretic protocols included a negative control, and as necessary, a positive control, and a molecular weight ladder. PCR bands were excised, and centnfiiged for 10 minutes at 5000 rpm.
Genetic variation was assessed through manual sequencing of approximately 400 bp using a nested vireo-specific primer, VireoH (Colette Baril and Madene Waker, unpubl.) (Table 3). Sequencing reactions were prepared according to manufacturer's instructions for Thermosequenase (Amersham Life
Science). Briefly the protocol is as follows. Each sequencing reaction ber sample) contains 2.0 pl reaction buffer; 5.0 pi &O; 1.0 pl primer; 2.0 pl Thenno Sequenase- DNA polymerase; and 10.0 pl
DNA. Termination mixes were prepared ('Gy,'A', 'T', 'C') containing 2.25 pl per sample. In the radioactive area 0.25 pl radioisotope (A,C,G,T) per sample was added to each tube of temination mix Materials and Methods: Genetic Analysis 26
Table 3. Primer sequences " used in the study of Vireo crassirostris
and congeneric species.
Primer Name Primer Sequence PCR Primers LPRO (5' to 3') GCTCCCAAAGCTGGTATTTT 16500 (3' to 5)' AGACCAAGGAGCCAGTCCCG Sequencing Primer I VueoH (5' to 3') TGAGTAGCTCGGTTCTCGTG I
a LPRO (Paton, unpubl.); 16500 Quhm (1992); VireoH (Baril and Wdker, unpubl.) Materials and Methods: Genetic Analysis 27
ND5 Cytochrome b tRNA tRNA tRNA Control Region tRNA -2000 -1 100 bp Thr Pro Glu -1227 bp Phe
Fig. 3. Schematic representation of mitochondrial geues in the vicinity of the control region and locations of primers used in the study of the Thick-biiied Vireo (Vkeocrassirosrris). Not to scale. Note that in birds ND6 is near the control region in conlrast to itç location bordering cytochrome b in rnammals (Quinn and Wilson 1993). Materials and Methods: Genetic Analysis 28
respectively followed by an alloquot of 2-50 pi into 4 tubes (A, C, G, T) for each sample. To each tube,
4.4 pi of the DNA template/polymerase mixture was added, followed by a drop of oil. Sarnples were
heated for 2 minutes at 95 OC then sequenced through a thennal cycle of 95 OC for 30 s, 55 OC for 30 s,
and 72°C for 60 s, repeated 29 times. On completion 4.0 pl of stop solution was added to each tube.
Samples were fiozen and kept with radioactive materials until ready for loading ont0 a sequencing gel at
which time they were heated to 90°C on a heating block. In order to obtah approxirnately 400 base
pairs of sequence, both a short (= 2 hours) and a long (= 5 hours) run were required. Population
sequences were obtained in one direction only.
Oddities arising fiom both amplification and sequencing reactions were a source of çoncern because it
was uncertain whether or not a nuclear homolog (Kidd and Fnesen 1998) had been amplified instead of
a mitochondrial copy. The problem took the fom of the presence of extra bands evident on the
autoradiographs. Inconsistencies in positive results fiom PCR also could have been the result of the
vireo-specific primers being designed off a nuclear copy. in order to resolve this issue a test was done
using a long template (2727 bp) derived fiom amplification with prirners located in cytochrome b and in
12S, providing a long template which was used in another PCR reaction with vireo-specific primers, and
with total MtDNA as a positive control (previously sequenced). The resulting sequence was compared to
the sequence fiom total DNA confinning its mitochondrial origin.
On completion of the sequencing reaction, sequences were read and input ont0 ESEE3s, Version 3.0
(Cabot 1994). Divergence between sequences was calculated with ESEEJs or Mega (Kumar et al. Materials and Methods: Genetic Andysis 29
1993). Nucleotide substitutions and nucleotide composition were analyzed, and haplotypes identified using ESEE3s and Mega. UPGMA and neighbor-joining trees were constructeci using the Jukes-Cantor
(1969) distance algorithm. Maximum parsirnony couid not be used as there were fewer phylogenetically informative sites than OTU's (Operationai Taxonomic Units) because overail sarnple sues are sinall and the sequences are too short in length. UPGMA and neighbor-joining are both distance methods, with the former assuming the operation of a rnolecular clock. Maximum parsimony, using only informative sites, assumes that evolution proceeds by some Ieast action principle with the fewest changes possible from a given ancestral sequence to a descendent sequence. While neighbour-joining and UPGMA were performed on the genetic data, the high degree of haploîype sharing in the Bahamian archipelago populations made these analyses confusing for interpretation. instead the 'minimum spanning tree' was constnicted with the most parsimonious connections among haplotypes illustrating haplotype shaiing and population structuring (Crandall and Templeton 1993). A relative rate test was not performed on this data set because the divergence thne is too recent. Divergence time between Vireo crassirostris and
K griseus was calculated using a rate of 20.8% per one million years for control region, domain 1
(Baker adMarshall 1997).
Control region sequence data were anaiyzed for Vireo crassirosfrisand congenerics (V: piseus, K pallens and K nanus) using UPGMA. Pairwise genetic distances were cdculated according to Kimura's
(1 980) two-parameter rnodel. Bootstrap tests (1000)replicates were run to assess the robustness of the clusters identified. Materials and Methods: Genetic Anaiysis 30
Genetic heterogmeity within popdations was estimated by calculating haplotype diversity (h) fiorn the percentage fiequency of haplotypes (Williams and Benzie 1997) fiorn eight populations (Bahamas:
Abaco, Andros, New Providence, San Salvador; Cayrnan Islands: Grand Cayman, Cayman Brac; Turks and Caicos: Providenciales and MiddIe Caicos treated as one population; Cuba: Paredon Grande Cay).
Haplotype diversity is defined as
where n is the sample sue (the number of individuals in each population for haploid data), S is the number of haplotypes in the sample, and pk is the fiequency of the kh haplotype in the sample (Lynch
1996).
Nucleotide diversity (z) is a measure of polymorphisrn in a population calculated fiom the average number of nucleotide differenccs per site between any two randomly chosen sequences (Nei 1987).
Nucleotide diversity is calculated by Materials and Methods: Genetic Analysis 3 1
where pu is the estimatecl sequence divergence between the ith andjth haplotype (the proportion of base
pair substitutions ) and fi and fi are their fiequencies (Nei 1987). 1 calcdated the fiequencies of al1
haplotypes (see Results) and generated the proportion of nucleotide ciifferences (transitions only) using
Mega (Kumar et al. 1993).
Gene flow, or migration (N,) was calculated using the nurnber of rare alleles per popdation according
to Slaîkin (1985)
wherep(1) is the average fiequency of aileles unique to a population, and a = -0.505 and b = -2.440 are
empirically derived constants (see Lynch 1996). Estirnates of gene flow were adjusted by the ratio of 25
(Slalkin 1985).
Analjwis of hurricane data
Tropical cyclone data were analyzed fiom the National Hurricane Center, Miami, Florida and the
National Clirnatic Data Center, AshviIle, North Caroha. Permission to access these data over the internet was provided by Dr. Robert Sheets, Director, National Hunicane Center, Miami, Florida and
Neal Loft of the National Chnatic Data Center, Ashville, North Carolina. Based on the latitude and longitude of the Bahamas and Caymans, hurricane patterns for the last 120 years were sought. Direction, severity and fiequency of hurricanes in the region were of particular interest. Results: Bioacoustic
BIOACOUSTiC RESULTS
Audiospecfrographicanalysis
Eighty syllable types were identified in the eight Thick-billed Vireo populations. Of these, 32 syllable types are unique, that is they are each found only in one population. A syllable type is an individual sylfable and ail of its variants. The number of syllable types (memes of length 1) recorded in each population, and their corresponding nuinber of unique (private) syllables are show in Table 4. Syllable fiequencies range fiom 720 Hz to 13,760 Hz, and length ranges fiorn -01563to -6062 s (Andros Island)
(Fig. 4). The mean syllable duration is .1497 s, and mean minimum and maximum fiequencies are 2575
Hz and 5847 Hz, respectively. The mean overall percentage of high fiequency syllables (>8 Khz) relative to the total number of syllables is 19.07% and temporally long syllables (B0.3 s) is 14.47%
(Table 5).
In a given Song, intersyllabic intervals range fiom .O 1875 s to over .2 s, and are longer between the first two syllables and the last two syllables. intersyllabic intervals are the 'silent' thne elapsed between any two syllables within a Song type. Therefore, short songs of foudfive syllables have a higher mean intersyllabic interva1 than songs with more than foudfive syllables because fewer syllables are sung within the saine time period. The mean intersyllabic intervai for songs on Providenciales, Turks and
Caicos is .O9 s, however when short songs are removed fiom the calcuiations, the mean decreases to
.O6 S. This pattern is consistent across the populations. Therefore short and long songs are closer in overall length than might be expected because as songs include more syllables, intrasyllabic time decreases between the additional syllables. Results: Bioacoustic 33
Table 4. Number of syllable types (mernes of length 1) recorded and the corresponding number of
unique (private) syllables in each fireo crussirostris population. Asterisk (*) indicates merging of data
sets from two or more years.
( Population ( Numberof ( Nurnber of I 1 syllables (meme ( unique syllables
1 New Providence 1 33* 1 Andros Island 1 39 *
Abaco IsIat~d 41* 8 1 San Salvador I Providenciales and Middle Caicos, Caicos Islands, Turks and Caicos
( Grand Cayman
Cayman Brac 21 1
Paredon Grande l Cay, Cuba Fig. 4. The longest syllable (.6062 sa)as recorded for a Vireo crassitostris, Andms Island, Bahamas. Resuits: Bioacoustic 35
Table 5. Syllable measurements: minimum and maximum fiequency, maximum syllable length,
percentage high fkquency (> 8000 Hz.) and long syllabIes (> 0.3 seconds) in eight island populations
of Vireo crassirosZris.
1 Population 1 MUllmum 1 Maximum Syllable % High % Long fkequency fiequency duration fiequency duration andmean andmean Minimum, syllables syllables (Hz) (Hz) Maximum and (Hz) (> 0.3 (Mean) sec.) New 1O00 13,280 .O2 188 - .5344 23.25 13.95 1 Providence (2350) (5771) (. 1781) Andros 1240 13,760 .O250 - .6062 19.04 19.05 (245 1) (6088) (. 1777) Abaco 1080 12,320 .O 1563 -.5000 29.4 1 17.64 (2889) (6689) (. 11 1 1) San Salvador 1200 11,360 (2096) (5372) 1 I1 Grand Cayman 300U 10,240 .01563-.4219 13.33 3.33 (2259) (5000) (. 1236) Cayman Brac 720 10,800 .O1875 - -3094 17.85 0.05 (25 1O) (586 1) (. 1373) Providenciales, 1280 10,640 .O1563 - .3781 21.57 ' 15.69 Turks & Caicos (2746) (5800) (.1414) Paredon Grande 1320 11,120 -01563 - -4000 17.85 17.85 (Cuba) (3305) (6200) (.1517) Results: Bioacoustic 36
In the sharing of syllables among populations (Table 6), the highest percentage occurs behireen Abaco and Andros (38.75%). The five Bahamian archipelago populations (Abaco, Andros, New Providence,
San Salvador, and Turks and Caicos) when compareci in al1 combinations share fiom 23.75% - 38.75% of their syllables. The Cayman Island populations (Grand Cayman and Cayman Brac) show low levels of sharing within the Caymans and with the other populations (< 22 %).
In neighbor-joining (Fig. 5), UPGMA (Fig. 6) and maximum parsimony (Fig. 7) trees the Cayman
Islands form one cluster and the Bahamian archipelago populations (New Providence, Andros, Abaco,
San Salvador and Turks and Caicos) form a second cluster. in neighbor-joining and UPGMA trees, northem Bahamian islands group together (Andros, Abaco and New Providence). In maximum parsirnony New Providence clusters with San Salvador and Turks and Caicos in the shortest tree, but is unresolved in the bootstrap analysis. The relaîionship of the Paredon Grande Cay (PGC) population with the other populations of Thick-billed Vireos varies arnong the three tree-building rnethods. In the neighbour-joining tree PGC clusters with the Bahamian archipelago populations; in the UPGMA phenogram it is an outlier to al1 of the other populations; and in maximum parsimony it is also an outlier, and is unresolved when a bootsîrap analysis is pdormed. Technical problems in the field influenced recording quality in Paredon Grande Cay which may explain the variability in the trees. As expected Vireo crassirosrris approximans (a chatter singer) is an oudier in ail trees. Bootstrapping of the maximum parsimony tree produced no nodes greater than or equal to 95% confidence; however,
Abaco and Andros had a confidence level of 92%.
Neighbor-joining and UPGMA methods produced phenograms showing three distinct clusters: the
'Capan' cluster (Grand Cayrnan and Cayman Brac); the 'Bahama' platform cluster (Abaco, Andros Results: Bioacoustic 37
Table 6. Number of syllables (N = 80) and percentage of syllables (%) shared between populations of
Vireo crassiroslris .
Populations New Andros Abaco Providence TSalvador Cayman Grande, C T Brac 1 Cuba rAbaco San 1- 1- Salvador
Paredon
Turks and Caicos Results: Bioacoustic 38
; PGC
Fig. 5. A neighbor-joining tree based on Jaccard's coefficient showing level of syllable sharing among populations of the Thick-billed Vireo (Vireo crassirosfris) and Vireo crassirostris approximans (Isla
Providencia, Colombia) as outgroup. (AB = Abaco, AN = Andros, NP = New Providence, SS = San
Salvador; T & C = Turks and Caicos Islands; PGC = Paredon Grande Cay, Cuba; GC = Grand Cayrnan,
CB = Cayman Brac; PV = Isla Providencia, Colombia). AB, AN, and NP = 'Bahama' platform cluster;
SS and T&C = 'trench' duster; and GC and CB = 'Cayman' cluster. Results: Bioacoustic 39
Level AB AN NP SS T&C GC CB PGC PV Level
Fig. 6. UPGMA phenogram based on clustering of Jaccard's coefficients showhg level of syllable
sharing among populations of Vireo crassirostris with l? c. approxintans (Ida Providencia , Colombia) as outgroup. (AB = Abaco, AN = Andros, NP = New Providence, SS = San Salvador; T & C = Turks and Caicos Islands; PGC = Paredon Grande Cay, Cuba; GC = Grand Cayrnan, CB = Cayman Brac; PV
= Isla Providencia, Colombia). AB, AN, and NP = 'Bahama' platform cluster; SS and T&C = 'îrench' cluster; and GC and CB = 'Cayrnan' cluster. Results: Bioacoustic 40
PV AB AN NP SS T&C GC CB PGC
Fig. 7. Shortest maximum parsimony tree (length = 83 steps; Consistency index (Cl) = 0.530; Retention index (RI) = 0.473) showing level of syllable sharing arnong populations of the Thick-billed Vireo
(Vireo crussirastris) with P! c. approximans (Isla Providencia, Colombia) as outgroup. Heuristic search, branch and bound and exhaustive methods dl produced the sarne tree. Bootstrap values of 1000 replicates is shown. (AB= Abaco, AN = Andros, NP = New Providence, SS = San Salvador; T & C =
Turks and Caicos Islands; PGC = Paredon Grande Cay, Cuba; GC = Grand Cayrnan, CB = Cayrnan
Brac; PV = Ida Providencia, Colombia). Results: Bioacoustic 41
and New Providence), and the 'trench' cluster (San Salvador, Turks and Caicos) - birds on islands separated by deep sea trenches at least throughout the Pleistoscene, and probably never interconnecteci
(Buden 1987). These trees are consistent with the biogeography of the region with the northem
Bahamian islands clusterhg together; the more distant Bahamian archipelago islands of San Salvador and the Turks and Caicos clustering together; and the Cayman populations, being the most geographicdly rernote, clustering together.
When the 'Bahama' platform and 'trench' clusters are pooled, dltrees (neighbour-joining, UPGMA, and maximum parsimony) are characterized by a Bahamian archipelago cluster which al1 island-pairs therein share at least 23% of their syllables. The cophenetic correlation coefficients of 0.83 (neighbour- joining) and 0.96 (UPGMA) give a good and excellent representation, respectively, of the original data matrix of Jaccard's CO-efficents.
Meme diversity as rneasured by the effetive number of memes (se) is highest for island populations in the Bahamian archipelago, and lowest for the more rernote Cayrnan Islands (TabIe 7). Abaco, one of the Iargest islands, has the highest merne diversity; however, there is little overall difference in meme diversity among the populations.
Meme flow (Table 8) was corrected for sarnple size following Slatkin (1 985). The highest merne flow occurs in Abaco and Andros, the two largest islands inhabited by Vireo crassirostris and in geographic proximity to each other (100 km between closest points). These islands also share the highest percentage of syllables (38.7 %). See Table 6 for number and percentages of syllable sharing among populations. Results: Bioacoustic 42
Table 7. Meme identity and diversity within Vireo crassirosfrispopdations. Meme identity O),
unbiased estimate of meme identity ( Î ), effective nurnber of mernes (%) and unbiased estimate of
effective memes ( s, ) are shown. Diversity masures were not caIculated for Paredon Grande Cay
(Cuba) as individual incidence of memes is not available.
A
Population 1 i se & Bahamas New Providence .O59 1 .O526 16.92 19.01 Andros -0503 .043 8 19.88 22.83 Abaco .O414 .O346 24.15 28.90 San Salvador -0560 .O495 17.86 20.20
Turks and Caicos Providenciales and .Middle Caicos 0.0494 .O427 20.24 23.42
Cayrnan Islands Grand Cayrnan 0.0778 -0683 12.85 14.64
- - -Cayman Brac 0.0677 .O527 14.77 18.97 - Pardon Grande Cay, Cuba Results: Bioacoustic 43
Table 8. Meme flow values (Na)for seven populations of Vireo crmsirostris
(Paredon Grande Cay not available).
Population Meme Flow
San Salvador 1 1.75 Grand Cayman 0.74 Cayrnan Brac 1 1.5 3 Providenciales 8.60 and Middle Caicos, Turks and Caicos Results: Bioacoustic 44
Song type analysis
The Thick-billed Vireo demonstrates a high degree of Song plasticity with an extensive repertoire of
Song types and variations. Song types range fiom 0.4625 - 2.775 seconds in length and consist of fiom 3 to 14 syilables with most songs consisting of 5 to 8 sylables (Table 9). The populations of the northem Bahamas have the highest number of song types which is consistent with their high meme diversity (New Providence, Andros and Abaco), and high meme flow (~bacoand Andros).
A range of combinations and permutations of song types have been identifid in the meme pool of each population, but few Song types cm be temed as typically representative of the species. Songs are terminated in a variety of ways, including chips (low or high fkquency) and buzzy syllables.
Recornbinations are evident in the construction of individual songs, with syllables of previously uttered
Song types included in new Song types. The identifiable sound of the species takes the fom of a non- musical buzzy, buny syllable type which is incorporated uito the majority of Song types. This syllable type is fiequency modulateci and Sung at varying fiequemies and syilable lengths. The singïng of a group of syllables of one syllable type (chip note repeated three times) was Sung in the context of different song types by bùds on Andros, New Providence and Abaco. This pattern was noted in both
1996 and 1970's analyses.
In general, sharing of song types was not found between populations, with the following exceptions
(Figure 8a - 8c). Song type # 19 fiom Andros Island is sùnilar to Song type # 1 fiom Abaco (Fig. 8a),
Song type #4 from Abaco is similar to song type #17 fiom New Providence (Fig. 8b), and Song type #11 fiom New Providence (1966) is similar to song type #6 fiom Abaco (Fig. 8c). Table 9. Populations of Vireo crassirostris sarnpled in the analysis of number of song types per
population and number of song types shed by at Ieast two individuals.
Min. and Min. and max. Highest # ol Sharing of birds max. # of song length song types song types syllables (seconds)(mem record4 foi within a per Song in brackets) individual population bird by at leasî two birds
Cuba
Providenciales and Middle Caicos, Turks and Caiws 8. Andros Abzlco
8b. New Rovidence Abaco
8c. New Providence Abaco
Fig. 8. Similarity in Song types among Vireo crassirostris populations. Song type # 19 fiom Andros is similar to Song type #1 fiom Abaco (Fig. 8a); çong type #4 fiom Abaco is similar to Song type # 17 of
New Providence f Fig. 8b); and Song type # 1 1 6om New Providence (1966) is similar to Song type #6 fkom Abaco (Fig. 8c). Results: Bioacoustic 47
Within popdation sharing of song types was noted (Table 9). Among 12 individuals recorded on
Providenciales, there were 28 song types plus variations. Only Song type #3, recorded by Peck and me
(June 1997) including its variations were Sung by more than two birds (N = 5). Four other song types
were each sung by two individuals, while the rernaining Song types (N = 23) were each found in one
individual only.
Shm-ing of song types within a population across time was noted only once in this study. Song type #7 occmed in 1971 and 1997 on Grand Cayman (Figure 9).
Visuai assessrnent indicates that Vireo crassirostris has a repertoire of at least 13 (Paredon Grande Cay) to 4 1 (Andros) song types in each population. No two birds sang exactIy the sarne song, even on the same island, although some sharing of Song types witliin a population was seen. High intraspecific and intra-population variation in Song iypes was found. hdividd birds may sing at least nine song types at a given time and probably many more. To record the full repertoire of an individual it would be necessary to record a bird continuously throughout its lifetime. The l3O+ birds were represented by over
243 Song types plus variations.
Individual birds sing a variety of song types and variations of each, incorporahg many different sy1Iables. Each bird has its own repertoire, switching song types fiequently and generaily singing Song types different fiom a neighbour. In a repertoire of six different Song types by a New Providence thick- bill recorded by Barlow (July 1971), the bird alternaîed between Song Type 'a' and Song Type 'b'
(baababaab) and then 'abandoning' those two, changed to two 'new' Song types, c and d (Figure 10).
This type of song switching is well known in scmb Weos (Barlow, in Litt.). Fig. 9. Temporal sharlig of song types by chronological populations of Vireo crassimstrir recorded on
Grand Cayman. Song type #7 was recorded both in 197 1 and 1997. Song type a Song type b
Song type d
Fig. 10. A mix of song types within a singing bout by a Vïreo crassirosfris recorded on New
Providence. Song type a and song type b alternated, followed by altemation of song type c and song type d. Results: Bioacoustic 50
A male recorded on Providenciales, Turks and Caicos (18 June, 1997) demonstrates the chronologicd change fiom one Song type to another song type (Figure 11). Nine different Song types plus variations were recorded within 20 minutes. Song type #5 altemated with song type #6; followed by a variation of
Song type #6; then song type #7 was followed by a variation of song type #7 including an additional syllable resernbling tlie long buzzy syllable fiom song type #6 which preceded it.
Long song or subsong (a seemingly randorn assemblage of syllables with no set pattern or rhythm, generally spaced equidistantIy fiom each other in time) has a disproportionately high nurnber of high frequency syllables (15-53 %) in this species and may last over 8 seconds. Inter-syllabic duration in this type of singing is longer ( > .25 sec) than in a song type, however recognizable song types may be included in these songs. The thick-bills heard by P.W. (Bill) Smith (in Litt.) in south-eastern Florida
(September 1989) were reported to sing a rarnbling song. Results: Bioacoustic 5 1
Song type fi6
Song type #7 variation
Fig. 1 1. Chronologicai development of song types in a Vireo crassirostris recorded on Providenciales
(18 June, 1997) demonstrating one way that Song types are changed after one song type has been uttered
25 to 50 times. Results: Genetic
GENETIC RESULTS
IntraspeciJic variab ility
Within the contiguous 389 base pairs of the control region (Appendix 3) which I compared, conserved
seqiience blocks are evident as reported by other investigators (Marshall and Baker 1997; Kidd and
Fnesen 1998). Sequencing fkom the 5' end of the region shows a relatively wnserved block (1- 124 bp),
followed by a hypervariable region (125- 163 bp) and flanked by another conserved sequence block
(164 - 389 bp). Comparison of the 45 Yireo crassirostris sequences reveds substitutions at 22 positions, al1 of which are transitions. There are no deletions or insertions. These sequences are represented by 17 haplotypes (Table 10, Fig. 12) with hapfotype frequencies shown in Table 1 1. Of these, 13 haplotypes are found in the northern Bahamas and Paredon Grande Cay (Cuba). The sample fiom Paredon Grande
(Cuba) is small (N = 2) but this small breeding population has the cornmonest haplotype found in the northern Bahamas (haplotype # 1). Birds sampled fi-om the Turks and Caicos and the Cayman IsIands show population structuring. Of the 12 birds sampled fiom îhe Turks and Caicos, I O are from
Providenciales (FI= 10) and 2 are fkom Middle Caicos (N=2). Of these 1 1 share haplotype # 16 and 1 has haplotype #17, which resembles haplotype #16 but with one additional substitution. The two birds fiom
MiddIe Caicos me represented by haplotype #16. The neighbor-joining tree is included for comparative purposes (Fig. 13) with the White-eyed Vireo (Kgriseus). Results: Genetic 53
Table 10. Haplotype designation and variable nucleotide positions (L-strand) in 17 Vireo crassirosfris mtDNA control region sequence hapIoîypes fiom 8 popullations. Haplotypes are numbered 1 - 17.
Variable nucleotide position numbers refer to the 22 sequence positions of nucleotide substitution within
389 base pairs sequenced. Haplotype 1 is the reference sequence. A change in nucleotide fioin
Haplotype 1 is indicated by a different nucleotide; sequence identity is indicated by a dot ( . ).
(AB = Abaco, AN = Andros, NP = New Providence, SS = San SaIvador; T & C = Turks and Caicos
IsIands; PGC = Paredon Grande Cay, Cuba; GC = Grand Cayman, CB = Cayman Brac). Variable positions for each of the 45 Vireo crassirosfrissequences are shown in Appendix 4.
Variable Position Population
1111111 1992334555 2125792679 GACTCGCATC ATGAACTTTC NP,AB, SS, PGC G...... CT NP G.. ..T.... NP .....A.G...... C.. NP ...... C.. NP,AN,AB A...... *.....C... SS ...... C... SS ....T.T... SS ....T..... AN G...... AN AN .....A.... AN .....T.... A3 GC CB ...C.A...... G...... T&C ...C.A...... G...... T&C Results: Genetic 54
Table 1 1. Percentage fiequency of haplotypes identifieci in eight populations of Vire0 crassirosfris.
Haplotype # refers to the haplotypes shown in Table 10. (n) is sample size.
Hap # New Andros Abaco San Turks Grand Cayman Paredon Providence SaIvador and Cayman Brac Grande Caicos C~Y Results: Genetic 55
Fig. 12. Minimum sp&g tree showing the relationship of haplotypes in eight populations of Vireo crassirosfris. Numbers in circles represent haplotypes (1 -17). Cross bars on branches equal the nurnber of transitional substitutions between haplotypes (there are no transversions). Circle sizes are consistent with fiequency ofhaplotype; the maIIest circles represent only one individual; the largest circles, haplotypes land 16 represent 10 birds and 11 birds, respectively. Abbreviations are as follows:
Bahamas: NP=New Providence, AN=Andros, AB=Abaco, SS=San Salvador; Cayman Islands:
GC=Grand Cayman, CB=Cayman Brac; T&C = Turks and Caicos; PG =Paredon Grande Cay (Cuba). Results: Genetic 56
Fig. 13. Neighbour-joining tree showing geneaiogical relationships arnong control region haploîypes in
eight populations of the Thick-billed Vireo (Vireo crussirastris) with the White-eyed Vireo (K griseus)
used as the outgroup taxon. Collection locale of each bird is abbreviated to the right of each specimen
number (AB = Abaco, AN = Andros, NP = New Providence, SS = San Salvador; T & C = Turks and
Caicos Islands; PGC = Paredon Grande Cay, Cuba; GC = Grand Cayman, CB = Cayman Brac). Tic :mno51 TCC Results: Genetic 58
Within-site haplotypic diversity (A)ranged fiom 0.0 (Grand Cayman, Cayman Brac, Paredon Grande
Cay) to 0.905 (Andros) and nucleotide diversity (R)ranged fiom O (Grand Cayman, Cayman Brac,
Paredon Grande Cay) to 0.005 (Andros) (Table 12). Low vaIues of diversity for the Turks and Caicos
population (h = 0.167 and ~t= .0002) are evidence of population structuring.
Gene flow (N,) is highest (Table 13) for the northern Bahamas (N, = 3.7545) (New Providence, Andros,
Abaco and San Salvador). The Turks and Caicos population of thick-bills has low gene flow
(N, = .0654) consistent with its Iow haplotypic and nucleotide diversity. When the Turks and Caicos
population is included with the northem Bahamas to comprise the Bahamian archipelago, the estimate of
gene flow diminishes (N, = 2.0283). Sample sizes for the Cayman and Paredon Grande Cay
populations were too srnall to calculate gene flow.
Nucleotide composition (Appendix 5) is AT rich and low in G (Domain I), consistent with low
functiond consîraints in the non-coding control region. Overall base composition for 389 base pairs
shows a paucity of G (1 5.9%) of the light sîmnd; cornpared to about 28% for each of the other bases.
These findings are in accord with Baker and Marshall (1997) who reported similar composition
(Domain i) in nine avian species represented by four families (three Anatidae, two Scolopacidae, two
Phasianidae and two Fringillidae). Results: Genetic 59
Table 12. Haploîype diversity (h) and nucleutide diversity (x) within
eight populations of Vireo crassirosfris.
Population 1 (W 1 (70 1 Bahamas 1 New Prov 0.857 -0049 Andros 1 0.905 1 .O05 1 Abaco 0.833 -0022 San Salvador 0.633 .O022
Paredon Grande Cay (Cuba) O O
Cayman Islands Grand Cayrnan O O Cayman Brac O O
Turks & Caicos Providenciales O. 167 .O002 Results: Genetic 60
Table 13. Estimates of gene flow (N,) in populations of Vireo crassirosiris in the northern Bahamas",
Turks and Caicos Islands and for the Baharnian archipelagob.
1 Northem Bahamas 1 3.7545 1 I Turks and Caicos -0654
a. Northern Bahamas include New Providence, Andros, Abaco and San Salvador.
b. Bahamian archipelago includes al1 populations of the nortliem Bahamas plus the Turks and Caicos
population. Results: Genetic 61
Phenetic analysis of Vireo crassirosîris and congeneric vireos
Sequencing of 389 base pairs of conîrol region for K g. griseus, yielded nine substitutions, dl
transitions. In cornparison to the most comrnon haplotype among Thick-billed Vireo populations, this
represents a 2.3 % divergence suggesting a divergence time of approximately 1 10,000 ybp. Cytochrome
b analysis (Barlow and Peck 1996, unpubl.) yields a 1.1 % divergence (274bp) indicaihg a
divergence time of 250,000-500,000years. While seemingiy contradictory, divergence times should be seen as estirnates because mitochondnal DNA yields diverse rates of evolution that vary among taxa, across gene regions and within and between genes (Mindel1 et al. 1997).
Two control region sequences fiom the Flat-billed Vireo (V: nanus) fiom Hispaniola show an 1 1.5% divergence ftom K griseus, consistent with an ancient origin of the sub-genus Yireo in the region whife the Mangrove Vireo (K pallens) reflects a divergence at approximately the 5% level. A
UPGMA phenogram with Kirnura (1980) two-parameter correction shows the relationships among
Yireo crassirosîris and congenerics (Fig. 14.) For the majority of nodes, bootstrapping produced values of greater than or equal to 98% confidence. Results: Genetic 62
1 I 6?+--- V. crassirostris 1
1 +---- V. pallens (Belize)
1 1 1 1 +------I + V. pallens (Mexico) 98+------1 99+ V. pallens (Mexico) +- V. nanus
Scale: each - is approximately equal to the distance of 0.001107
Fig. 14. UPGMA phenogram with Kirnura two-parameter correction of control region sequences of
Vireo crassirosiris and congeneric vireos: White-eyed Vireo (K griseus), Mangrove Virexi (K palfens) and Flat-billed Vireo (K rîanus). Bootstrapping of 1000 rqlicates is shown on branches. Results: Humcane activity
Hurricane activity in the West lndies
Based on the latitude and longitude of the Bahamas and Caymans, hurricane patterns for the last 120 years were accessed (Appendix 6). The Bahama Islands and their banks lie between 19O 30'N to 27"
30'N and 6S0 30'W to 8 1 3 1'W (Buden 1987). While the total area of the banks is ca 124,716 km2, exposed land is ody 1 1,406 km2 (Buden 1987). Hmicanes have only been tmcked since 1875, therefore historical trends are impossible to determine. Despite this caveat, the available data show that hunicanes in the region of the Bahamian archipelago (Bahamas aad Turks and Caicos Islands) tend to move fiom north to south, and more fiequentiy and forcefidly hit the more northem islands of the
Bahamas. Hilumcanes may have had a more significant impact on mixing of the northem Bahamian avifauna than for the avifauna of the southem isIands. In the 100 years fiom 1875 - 1975, Buden (1 987) determined that at lest 35 hmicanes passed over Cuba, while another 35 crossed over another neighbouring land rnass prior to staging destruction on the Bahamas.
The Cayman Islands lie between 19O 20'N and 19O 43'N and 79' 50' W - 81 O 2 1' W. While hurricanes are Iess fiequent in the Caymans, when the islands are hit, the impact can be severe, as in 1944 and again in 1996 when Hurricane Gilbert wreaked destruction. Discussion
DISCUSSION
Song type variability consistent with island condilions
There is no such thing as the sorig of a species (Hartshorne 1973). This is particularly true for Song in
the Thick-billed Vireo. A high degree of variation is demonstrated at al1 levels; witliin and between
individuds in a population, within and between populations, and chronologicaUy within a population.
Notably, during the course of my research, several peopIe fiom different islands have spontaneously and
accurately comrnented th& 'their' thick-bills sound different than thick-bills on other islands. My
research supports these casud observations.
The acoustic environment (in tenns of the number of çonspecifics, number of birds of other species
singing, quality of Song and position of songsters in the ecological setting) plays a role in the acquisition of memes and subsequent repertoire thaî an individual develops. Marler (1 960) hypothesized that an
inverse relationship exists between avifaund contplexity and vocal cornplexity of a given species. In a species-rich mainland environment, where acoustic inter-specific competition is high for that 'space', con-specifics must maintain a repertoire that is relatively circumscribed. This unique species signature is necessto acquire territory and attract mates. Species on songbird-depauperaîe islands, however, have less acousticai competition and a smaller listening audience, and are therefore fieer to improvise. Lynch and Baker (1993) in a study of Chaffinch Song on the mainland and islands found that Song variability is higher in peripheral isIand populations. Island conditions with their depauperate avifauna encourage a Discussion 65
relaxation in species distinctiveness resulting in increased song variability and less pressure to have
'distinctive' songs (Marler 1960, Lynch and Baker 1993). The 'loss of contrast' hypothesis (Thielcke
1973) refers to the loss of species-specific ciifferences in song in the absence of coexisting related
species. During the breeding season, the islands within the range of Vireo crussirosfrisare generally
song-bird depauperate. In my study populations, the only resident sympatric Weos are the Black-
whiskered Vireo (V: altiloquus) on Cayman Brac and on the Bahamian archipelago islands (except San
Salvador); and the Yucatan Vireo (K magisfer)on Grand Cayman. Notably these arboreal vireos in the
sub-genus Vireosylva (eye-line vireos), usually forage in higher vegetation than scmb vireos in the sub-
genus Vireo. This niche differentiation is the same for sirnilar habitat on the mainiand (Barlow 1980).
The loose syntax of song and extensive Song type variability in thick-bill populations are consistent with
the 'loss of çontrast' hypothesis. The combination and permutation of syllables yield an infinite number
of song types in each population in accord with the infinite aileles mode1 which projects that any Song type arising in a population is a new Song type, not previously found in the population.
Kroodsma (1 985) in his study of the Bewick's Wren (Thryomanes bewickii) found that island songs were higher in Grequency than their mainland counterparts indicating that fiequency range expands in the
absence of competing sounds. My results of maximum syliable fiequency agree with this finding.
Although thick-bills cannot be compared in the sarne manner as they are resnicted to islands, a cornparison of maximum fiequency with the White-eyed Vireo, its closest mainland neighbour
(phylogenetically), shows a higher fiequency range in the thick-bill(> 13 Khz) compared to 8.5 Khz in the white-eye (Borror 1987). If fiequency range of syllables increases, then it is conceivable that the Discussion 66
number of high fiequency syllables uttered in the absence of competing high fiequency sounds will increase to fi11 the gap. Tubaro and Segura (1 995) found that low latitudes and smailer body size
(Bergmann's Rule) cornelateci with higher maximum fiequacies in the Song of the Rufous-browed
Peppershrike (Cyclurhis pjanensis).
Isolated populations of a sedentary species becorne differentiated fioin each other as a fünction of both geographic distance and the length of lime of isolation. Because cultural evolution proceeds more rapidly than genetic evolution, didecticd changes in birdsong may be apparent to a trained listener within a hwnan lifetime. In contrast, micro-evolutionary changes at the genetic level are much slower, with genetic mutations occurring at a rate of one nucleotide change in thousands of years. Cavalli-Sforza and Feldman (1 98 1) note that in hurnan language, similarities cm disappear after 10,000 years of separation. Barlow et ai. (data 1998) found that 15% of the syllables recorded fkom two Black-capped
Vireos (V. atricapilhs) in Big Bend National Park (BBNP)persisted 30 years later. Among three populations of the sarne species in Oklahoma, syllable sharing was minimal as the population in the smallest site was extirpated during the course of the study, and the other sites comprising banded birds differed fiom each other as a fiction of philopatry and isolation througli habitat loss (Barlow, in Litt.).
Loss of song eIements is due to mutation, migration and random drift. As loss of individual memes occurs, Song type drift follows. Since song types naturally depend on their constituent elements, the loss of those elements will lead to dialecticd change. For example, two syllables Gcom song type #11 fiom
New Providence (Corneli Laboratory of OmithoIog~,data 1966) cluster together in the same marner as in Song type #6 fiom Abaco (1996). This cluster was not found in the 1996 recordings fiom New Discussion 67
Providence, dtbough the two syllables were retained, suggesting that the song type was either lost in the
intmening thrty years, or that it was present but not recorded in the 1996 population.
Temporal comparison of Song types within a population (ResuIts: Bioacoustic) yielded little similarïty
within intervening years (comparative data for New Providence, Andros, Grand Cayman). The one
exception was one song type on Grand Cayman found in both 1974 and 1997. This song type has either
been retained in the population or is a randoin recombination (Lynch, in Litt.) which because meme
diversity is low may have been reproduced. Retention of the Song type may be the result of a long-lived
individual (Ince et al. 1980) having an acoustical influence through several generations.
To a trained listener, switches fiom song type to song type, and even variations of song types can be
identified in the field, but it is in the sound laboratory where composition, mernory and pattern can be more fùlly appreciated. Upon analysis, sounds that sound alike in the field, may actually be found to dinèr structuralIy. The Thick-bilted Vireo generates a large number of song types, often incorporating syllables fiom preceding Song types into another song type. This non-random utilization of syllables has aiso been found in Cardinals (Cardinalis cardinalis) (Lemon 1975). The role memory plays is important, as a Song is not categorized as a song type unless it is a repeated arrangement of syllables that can be recognized as a unique pattern, according to my definition (Materials and Methods : Bioacoustic analysis). Often this repetition is sequential, while at other bmes a song type may be alternately sung with another song type, This repetition, dong with the interspersai of another song type demonstrates that the song was sung deliberately. In other words, even if the original arrangement of syllables was sung by chance, the repetition afYords it the status of a Song type. While a number of song types exist in Discussion 68
a population at a given the, they are recycled and continually evolving through the same mechanisms
that influence the loss and changing of song elements, that is, through mutation, migration and random
drift.
This anaiysis has shown that there is a continuum fiom one Song type to another song type with
variations in between. Along this chronologicai continuum, variations at one end will be more like the
earlier song type, while variations at the other end resemble the Iatter Song type. At some arbitrary point
determhed by visual gestalt by the researcher, a new Song type has been identifiai. The high degree of
variation and recombination evident in the song of Vireo crussirosfris, and characteristic of scrub vireos,
(Barlow, in Litt.), is consistent with songbird-depauperate islands and reduced avifaunal complexity.
Cornparison of the songs of Vireo crassirosfris and Vireo griseus
My bioacoustic results for the Thick-billed Vireo resemble findings discussed by R. A. Bradley (1980,
198 1) and Borror (1987) in their studies of song in the White-eyed Vireo (K griseus). The shape,
structure and organization of syllables of the two species are similar which may be evidence of wmon
ancestry. Borror (1987) found that White-eyed Vireos have between 11.7 song types and 15.3
variations. My observations suggest that individual thick-bills have at least 9 song types plus variations.
Thick-billed Vireos have both lower and higlier fiequency syllables than the 1.6 kHz to 8.5 kHz
fiequencies noted by Borror (1987) in white-eyes. In addition, the Tluck-biHed Vireo has a higher
percentage of long duration syllables (20.3 sec.) in the repertoire of each population (>13%) (except for the Caymaii Islands), compareci to the White-eyed Vireo (5%). The greater number of long syllables is Discussion 69
reflected in the longer songs of the thick-bill (x = 1.20 s), compared to the relatively shorter song of the
White-eyed Vireo (x = 1.02 s).
Hypotheses for wide-ranging dis[ributionof Vireo crassiroslris
Among the scrub-dweihg sedentary taxa in the sub-genus Vireo that fil1 a similar ecologicaI niche throughout the West indies, the Thick-billed Vireo is the only species that occws on so rnany islands
(Fig. 1). Al1 other island endernics of the sub-genus occur on one island, except for Vireo gundfachii which may occw on both Cuba 'proper' and on several islands off the south Coast of Cuba (Barlow, in
Litt.). ln contradistinction to the majority of these taxa which are highIy diverged fiom the ancestd
'white-eye' type, the Thick-billed Vireo is closely allied phylogenetically (2.3% diverged for MtdNA control region) to the White-eyed Vireo (K griseus) which winters in iow numbers throughout the range of the thick-bill.
Two models of diversification in the West Indies are relevant to this study. In one model, isolation on the large Greater Antilles (Cuba, Hispaniola, Puerto Rico and Jamaica) has resulted in the evolution of fiil1 species which are highly diverged fiom the White-eyed Vireo, for example the Cuban Vireo
(K gundfachii)of Cuba and the Flat-billed Vireo (V: nanus) of Hispaniola (Barlow, in Litt.). In the second model, Vireo crassiroslris, not liighly diverged fiom the White-eyed Vireo, is absent Erom the large islands of the Greater Antilles, but is widespread among small islands. There are no ancient vireos in the rage of Vireo crassirosiris, suggesting that the 'older' species in the sub-genus had Discussion 70
already been well established on 'the? islands before Vïreo crassirosfrisevolved. 1 postulate that
islands lacking a resident scrub-dwelIing vireo were the srnaller, flatter islands in the Bahamian
archipelago, which newly merged in the late Pleistoscene and therefore were available for colonization
by the Thick-billed Vueo.
The 'smdl island' distribution of the thick-bill involves a complex array of ecological, evolutionary and
biogeographical phenornena Faaborg (1985) in an analysis of ecological constraints on West Indian bird
distri butions found that guilds of gleaning insectivores include a large vireo, usual ly Vireo alfifoquus
(20 g), and a smdler warbler or vireo (1 I g). He found that the pattern held in Cuba with Vïreo gundluchii filling the 1 1 g niche. By inference, this might competitively exclude the Thick-billed Vireo, if it were not for the probability that the ancesid 'whiteeye' entered a new lodity for scrub vireos.
This environment was the emerging Bahamian archipelago which afforded the opporhuiity for a scrub vireo to colonize that area and to reach the Cayman Islands, by chance or through hunicane mediation, while it was still a powerfill fiyer. The thick-bill has clearly not lost its ability to disperse, as evidenced by its appearances in coastal Florida (Smith et al. 1990, Langridge 1988, Abramson 1974).
The cornbined effects of clhatic change and sea Ievel fluctuations is instrumental in the distribution of
West Indian taxa (PregilI and Olson 1981). During the late Pleistocene when arid conditions prevailed, scrub species had a selective advantage. Although Vireo crussirostris centres its activities in low-lying scrub habitat, it lias been observed foraging in vegetation of 1 to 6 metres and is found in a variety of available habitats. Murphy et al. (1 998) captured and banded thick-bilIs in scrub, mangrove, secondary growth and forest in San Salvador, Bahamas. Discussion 71
The distribution of the thick-bill fi& beginning stages of an allopairic model of speciation, wherein
distinct species may emerge with the diffitiation of 'initially-like' populations in geographic
isolation. Archipelagos, in particular, with their variety of islands and pattern of diversification, are
important in understanding the process of speciation (Grant and Grant 1996). The peripatric model,
which is a special case of the allopatnc model, may explain distributional patterns and diversification.
Mayr (1 982) proposes that a species cannot change sigiificantly in the central part of its range because
gene flow interacts to homogenize popdations, however, in peripheral isolates, where gene flow is
reduced, speciation or sub-speciation cm occur through founder effects. Anoiher mode1 postulates that
isolation is not a necessary condition sirice speciation rnay be initiated in allopatry, but continued and
completed in sympatry (Grant and Grant 1996, EndIer 1977). Hamilton (1 962) suggested that an inverse
relationship exists between the size of a geographic isolate and the degree of divergence associated with
that gene pool in isolation.
The allopatric distribution of Vireo crassirosrris is in accord with a recent evolutionary event, consistent with the presence of only transitional nuclwtide substitutions, the biogeography of the region and the
vocal and phenotypic sirnilarity between Vireo crassirosfris and Kreo griseus. The estimated time of divergence between the Thick-biUed and the Whiteeyed vireos is approximately 1 10,000 ybp using a rate of 20.8% per one million years for control region, domain I (Baker and Marshall 1997). This approximation is compatible with the northern Bahamas being submergeci c. 120,000 ybp (Morgan
1989) during the last interglacial. Altematively, thick-bills may have been in an island refigiurn during the rise in sea level at this time. Cment elevation of the Bahamas seldom exceeds 5 - 10 m, although there are a few ridges ,and hills that reach 30 - 60 m, with the highest point being 67 m on Cat Island
(Buden 1987). Grand Cayman and Little Cayman are low-lying, with elevations of 19 m and 13 m, Discussion 72
respectiveIy (P. E. Bradley 1995), while Cayman Brac has a maximum elevation of 44 m (144 feet).
High ground in the Bahamas, Cayman Brac and Cuba may have acted as refugia during the highest sea
levels, approximately 125,000 (Garrett and Gould 1984) and again 70,000 ybp (Pregill and Olson
1981). With the eventual Iowering of sea level, the species rnay have dispersed throughout the gradually
exposed archipelago.
Cultural and genetic analyses yielded paralle1 results for Thick-billed Vireo populations in the northern
Bahamas. This was demonsfrated by an apparent 'centre of eng'for 'crassiroslris' isolates çoming
into contact with one another across the, as evidenced by both gene and meme flow among these
islands. in contrast, the more geographically rremote populations in the Cayman Islands and in the Turks
and Caicos have become differentiated from the northem Bahama island populations. These results
are consistent with currently accepted taxonomy, with the Cayman population designated as Vireo
crassirostris alleni and the Turks and Caicos population named Vireo crassirostris stalagmium, while
îhe northern Bahamian populations retain the nominate form, Vireo crassiroslris crassirosfris.
The northern Bahamas have the highest meme diversity and meme Aow of al1 populations, consistent with the recent coalescence of some of îhese islands (New Providence, Andros and Abaco) 17,000 years ago and subsequent separation. San Salvador, to the south-east of these islands, and Providenciales
(Turks and Caicos), at the exberne southem end of the archipelago, have always been separated by deep sea trenches, and therefore never connected to another island. Therefore, in these islands, any meme flow inust have resulted fkom over-water stepping-stone dispersal through the islands or mediated by hurricanes. Geographic distances arnoiig the islands of the Bahamian archipelago are not great since over Discussion 73
700 isIands and cays are spread throughout the 950 km length of the archipelago. Available sample sizes
were too small to estimate within-population levels of gene flow for al1 populations; however, by
wmbining data it was possible to estimate levels of gene flow within the northem Bahamas and in the
Bahamian archipelago. Gene flow was highest for the islands of the northem Bahamas, consistent with
hypothesized 'mixing' of these populations as water levels alternately rose and fell, resulting in
emergent land and more islands.
Coalescence and separation have given the islands of the archipelago different biogeographical histones.
The geology of the continental islands of the northem Bahamas has served as a passive agent of
dispersal through coalescence and separation during the Pleistocene, while hwicanes have acted as
active agents with their direction, severity and fiequency impaçting the 'rnixing' of thick-bill
populations. Populations in the southern oceanic islands of the archipelago are undoubtedly influenced
by humicanes but the deep trenches separating the isIands likely precluded vicariance. Because thick-
bills may not demonstrate long distance dispersal, hurricanes have likely played a role in immigration and recolonization.
The inpacr ofhurricanes
The Bahamas are the target of fiequent hurricanes and tropical storms, with prevailing winds blowing fiom the east most of the year; fiom the south-southeast in summer and north-northeast in winter (Buden
1987). This suggests that hunicanes may have had a more significant impact on the rnixuig of the northem Bahamian populations of thick-bills. Kevel Lindsay (Internet 1998) reported that the recent arriva1 of the Green Iguana (iguana iguana) on Anguilla, after hunicanes Luis and Marilyn, is Discussion 74
contemporary proof thaî natural processes are important to the colonization and distribution of animals
in the West hdies.
Wunderle (1995)has studied avian responses to hwicane impact in the Caribbean. He concludes that
the ability to shift habitats confers a selective advantage on birds in this region. The indirect long-terni
effects of such violent storms are greater than the mortality directly caused by hi& winds and rainfall
during a hurricane. Frugivorous and/or canopy dwelling birds are more affected by loss of food, nest
sites and foraging substrates than insectivoraus, lower foraging species (Wiley and Wunderle 1993).
V: crassiroslris is ideaiiy suited to local conditions as a result of its preference for foraging in low, fast- growing deciduous scrub, and insectivorous/fhgivorous diet. With these strategies, the thick-bill may be adapted to persist in the aftermath of hurricanes. This is in contrast to the nectarivorous canopy dweIling
Bananaquit (Coerebaflaveola) whose populations were decimated on San Salvador, after Humkane Lily in 1996 (Murphy et al. 1998).
Origin of Cayman Thick-bilkd Vireos
What might be the origin of populations of Thick-billed Vireos on the Cayman Islands? Band (1 934) stated that "Grand Cayman has received most of its bird life from Jamaica and Cuba, whereas LittIe
Cayman and Cayman Brac have derived theirs fiom Cuba alone...". One hypothesis suggests that Cuba once harboured Vireo crassirosrris, which was subsequently competitively excluded by the Cuban Vireo
(V: gundlachii) (Garrido and Kirkconnell, in Lit, Wallace, in Litt.). Barlow (in Litt.) counters this Discussion 75
argument, indicating that based on its genetics and vocalizations Yireo gundlachii is a more 'ancient'
taxon than Vireo crassirostris or Vireo griseus, the latter of which winters in Cuba without competing
with Vireo gundlachii.
The geographic remoteness of the Cayman Islands dong with a Ievel of genetic divergence of 1% fiom
the most common thick-bill haplotype in the Bahamas, suggests that the Cayman colonizaiion resulted
fiom Bahamian archipelago 'founders'. Both genetic and cultural analyses show a higher level of
differentiaîion between Cayman Island thick-bills and other populations, which is consistent with the
rernoteness of these islands fiom other Thick-billed Vireo populations in the Baharnian archipelago, and
fiom each other (124 km apart). In contrast to the norihem Bahamas, the Cayman Islands have never
been joined. Birds fiom Grand Cayman and Cayman Brac, while sharing few sylIables (1 7.5%),
clustered together which is likely the result of retention of ancestral memes, convergence or very rare
dispersal events. Mme flow is conceivable but remote, particularly for Grand Cayman. Cayman Brac
has higher meme flow and diversity suggesting that it may have received potential immigrants due to its
position relative to Cuba, the Bahamian archipelago, Isle de la Tortue or Little Cayman. Dispersal by
hunicanes tlirough the Windward Passage between Cuba and Hispaniola may have been the conduit.
Cayrnan Brac would therefore likely be a stepping Stone for Grand Cayrnan colonization opportunities.
Faunder efect in the Turks and Caicos Islands
In contrast to the hi& degree of haplotypic sharing in the northern Bahamas (Andros, Abaco, New
Providence and San Salvador), the populations of Providenciales and Middle Caicos in the Turks and Discussion 76
Caicos are characterized by a predominant haplotype, not shed with any other population. Al1 but one individual (N=I 1) shared the same haplotype which was 0.8% diverged fiom the most comon hapfotype in the northern Bahamas, and one bird (N=l) had a haplotype with an additional transitional substitution. Although some of the shared haplotypes in the northern Bahamas were more than 0.8% diverged, they were found on several different islands as would be expected if populations came in contact fier an interval of separabon on separate islands. In cornparison, the Turks and Caicos birds were relatively homogeneous for the gene locus (control region) sequenced. My results suggest that this population at the southern extreme of the Bahamian archipeIago, represents a population that was established by a founder event or that it has undergone a genetic bottleneck.
While haplotypic diversity for the Turks and Caicos population is low, meme diversity is high. This high meme diversity dong with a high percentage of both long duration and high fiequency syllables is in accord with a loose syntax, consistent with a population which is a peripheral isolate. Both gene and meme flow were shown to be at low levels. Notwithstanding the low meme fiow, the population has a relatively high leve1 of meme diversity and a high level of syllable sharing with each of the other
Bahamian archipelago populations studied (> 26%). This suggests that founders came with a large repertoire of syllables, and in addition generated new syllables.
Codescent theory predicts that the oldest haplotype in a lineage should be the most wideIy distributed arnong populations (Takahata 1988) and it should also have the highest number of mutational connections to other haplotypes in the parsimony network (CrandaIl and Templeton 1993). Excoffier Discussion 77
and Langaney (1989) found that haplotypes of low fiequency are usually located at the 'tips' of a
network, while haplotypes of hi& frequency occur in the interior. The most common haplotype in the
Thick-billed Vireo populations is found in the interior of the minimum spanning m.As predicted by
coalescent theory, this haplotype has the most mutational derivatives and the widest distribution among
populations (Crandall and Templeton 1993).
Exarnining geographic patterns may be usefùl in inferring the topological position of a rare haplotype
(Crandall and Templeton 1993). Some Iiaplotypes have a high number of substitutions in comparison to
the cornmon haplotype, suggesting that intermediate haplotypes (Crandall and Templeton 1993,
Excoffier et al. 1992, Domelly and Tavaré 1986, Aquadro and Greenberg 1983) may be present either
in the population or in a neighbouring popuiation, particularly when a rare haplotype comprises too many substitutions to have hsenindependently. Also for unusud population structuring, such as
possible founder effects and genetic bottlenecks, one could infer the historic origin of the founders and whether the distinct haplotype is found elsewhere. In the case of minimal gene flow, O'Corry-Crowe et aI. (1997) note that a recent mutation is more likely found in~thesource popuiation than in a distant population. In this regard, supplementary data fiom the southern islands of the Bahamian archipelago, such as Mayaguana and Crooked Island (isolated by deep treiiches) would be useful in fùrther deteminhg speciation patterns and population structuring in the archipelago. Alternatively, some haplotypes may have been lost through stochastic lineage sorting, a self-pruning process which may through time elidnate a majority of luieages while some others persist (Avise 1994). Discussion 78
Bibby (1994) points out that over 25% of al1 birds have ranges of less than 50,000 km2,grouped
together into 22 1 Endernic Bird Areas, covering 5 % of the Earth's land surface. Therefore, the fùture of
a quarter of ail bird species depends on the success or failure of conservation efforts within this criticai
5 % ara (Bibby 1994). The range of fireo crassirostris fdls wiîhin this critical domain. In contrast to
mainland birds, species threatened in their small idand habitats may have less resiliency due to their
smail population size, habitat specificity and stochastic factors.
The scrub habitat on which Vireo crassirostris depends is a necessary condition for the survival of this
species. Cotgreave and Harvey (1994) describe the role of habitat type in measuring the evenness of
abundance in bird communities. They predict that simple habitats, like scmb, offer a limited nurnber of
large niches, in addition to several smaller niches, and th& species able to use large niches will be
abundant. The scrub in which K ct-assirosrris dwells is the key factor in terms of conservation. Scmb is
genaally not a valued commodity, aesthetically or economically. If scmb species are going to persist,
this habitat needs to be conserved.
The more restricted a population is geographically, the greater the impact upon its population in the
event of a catastrophic event, such as a hunicane (Simberloff 1994). Island species fdI within this
category, not only because of their lirnited range, but because these populations have already been reduced in numbers and range by habitat destruction (Simberloff 1994). Mangel and Tier (1994) emphasize that population viability analyses must take into account catastrophes, for without this Discussion 79
parameter, minimum viable popdation sizes will be too smaü. Although direct mortality is a factor in the loss of individual birds, species do not usually become extinct solely fiom the trauma of hurricanes, but may be extirpated in a poorly protected area in a catastrophic hurricane (but see Spiller et al. 19%).
The most important point is that in most accounts of extinctions of avian species, the ultimate cause of extinction is due to habitat loss, ofien human mediateci.
Vireo crassirm~rishas persisted for miIIennia and continues to be widespread on most islands. The real challenge to continueci persistence will be adaptation to human development, which brings with it increased habitat loss. The species appears to tolmte habitat fragmentation; it is the loss of habitat that will make it vulnerable. New Providence, in the Bahamas, home to many avian species, as well as the wintering grounds for migrants, has become a prime target for development because of its climate, beaches and economy. P. E. Bradley (1995) notes that a rapidly gowing tourist Uldustry in the Caymans has given rise to many hotels, and golf courses, with consequent habitat loss and fragmentation already contributing to a decline in bird diversity in the western part of Grand Cayman.
Yireo crassirostris has endured in the present century without any reai threat to its survival. Although natural threats always exisf it is the human-induced ones that pose a substantial threat to the long term survival of the species. By protecting scrub habitat, through decreasing habitat loss, this scrub dwelling species will continue to enhance the acoustical environment of songbird depauperate islands. The primary objective of this study has been.to determine patterns of divergence in cultural and genetic evolution in populations of the West Indian endemic Thick-billed Vireo. The hypothesis tested is that these sedentary island populations vqin th& level of divergence, culturally and genetically, as a fûnction of geographic isolation.
Specirographic assay of songs of 140 males recorded on eight islands comprisuig 80 syllables fomed three clusters: 'Cayman' cluster (Grand Cayman and Cayman Brac); 'Bahma' platform cluster (Abaco,
Andros and New Providence), and the 'trench' cluster - birds on isIands separated by deep sea trenches
(San Salvador and Turks and Caicos). The loose syntax of song and extensive Song type variability of the species is compatible with the 'loss of contrast' hypothesis in which selective pressure for species- distinctiveness is relaxed in a songbird depauperate island enviromnent.
The distribution of I&eo crassirostris is in accord with a recent evolutionary event consistent with the biogeography of the region and the estimated time of divergence between the Thick-billed Vireo and the
White-eyed Vireo, Weogriseus, approximately 1 10,000 years ago. The rnost significant event is thought to have been the coalescence and subsequent sepration of the northern Bahamas into the present array of islands, approximately 17,000 years ago. Genetic and bioacoustic analyses show pardlel patterns in levels of divergence in merne and haplotypic diversity with an apparent hi& level of mixing of these previously isolated populations.
The Cayman Islands, most distant f?om other Thick-billed Vireo populations, appears to have evolved largely independently. Their geographic remoteness, low level of syllable sharing compared with other Thick-billexi Vireo populations, and the hi& number of nucleotide substitutions relative to the most
common haplotype in the northern Bahamas, indicates that colonization of these islands resulted fiom
Bahamian archipelago 'founders' early on in the evolutionq history of the species. The population of
thick-bills in the Turks and Caicos Islands is genetically differentiated fiom the other Bahamian
archipelago populations with population structuring dso the probable result of a founder effect. In this
case, genetic and cdtural evolution are inversely relate4 evidenced by low haplotypic diversity but high
meme diversity. The levels of divergence show between populations of the Bahamas and the Turks and
Caicos Islands and Cayman Islands are consistent with the taxonomic classification of these populations
as separate races, V, c. alleni (Cory) and K c. stalagmium (Buden) respectively, as distinct fiom the
nominate form in the northern Bahamas.
The population of Thick-biHed Vireos, Vireo crassirostris approximans, of Providencia Island in the
SW Caribbean was used in the Song andysis as an outgroup because of its taxonomic position and
atypicd 'crassirosfris' Song. As expected, the popdation was distinct from al1 other Thick-billed Vireo
populations.
The geology of the continental islands of the northern Bahamas has served as a passive agent of
dispersai tbrougb codescence and separation during the Pleistocene, whiIe hurricanes have acted as
active agents with their direction, severity and fiequency impacting the 'mixing' of thick-bill
populations. Populations in the southern oceanic islands of the archipelago are undoubtedly influenced
by hurricanes but the deep trenches separating the islands likely precluded vicariance. Because thick- bills may not demonstrate long distance dispersai, hurricanes have likeIy played a role in immigration
and recolonization opportunities. Literature Cited
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Appendix 1. Example calculation of meme identity for the Thick-billed Vireo ushg the
population fiom Abaco Island, Bahamas. Meme identity is .0414.
I Mcme 1 Mcme occurrences 1 Frequcncy (F) of 1 Frqucncy (F~) pcr individual bird meme S 1 1 11143 =.O07 .O0005 S3 10 101143 1-07 .O05
S7 4 41143 = .O28 .O008
1 1 I 1 Total 1 143 1 1 Appendix 2 Specimens Used in Analysis Centre for Biodiversity, Royal Ontario Museum, Toronto, Ontario, Canada
Specimen Location Date of collection Collection #
Vireo crassirostris New Providence, Bahamas MRW 1 Vireo crassiroslris New Providence, Bahamas MRW 2 Vireo crassirosîris New Providence, Bahamas MRw3 Vireo crassirostris New Providence, Bahamas MRW4 Vireo crassirostris New Providence, Bahamas MRW 5 Vireo crassirostris New Providence, Bahamas MRW6 Vire0 crassirostris New Providence, Bahamas MRW 7 Vireo crassirostris San Salvador, Bahamas MRW 8 Vireo crassirostris San Salvador, Bahamas MRW 9 Vireo crassirostris San Salvador, Bahamas MRW 10 Vireo crassirostris San Salvador, Bahamas MRW II Vireo crassirostris San Salvador, Bahamas MRW 12 Vireo crassirostris San Salvador, Bahamas MRW 13 Vireo crassirosf ris San Salvador, Bahamas MRW 14 Vireo crassirostris San Salvador, Bahamas MRW 15 Vireo crassirostris San Salvador, Bahamas MRW 16 Vireo crassirostris San Salvador, Bahamas MRW27 Vireo crassirostris Andros, Bahamas MRW 18 Vireo crassivostris Andros, Bahamas MRW 19 Vireo crassirostris Andros, Bahamas MRW 20 Vireo crassiroslris Andros, Bahamas MRW21 Vireo crasszrostris Andros, Bahamas MRW 22 Vireo crassirostris Andros, Bahamas MRW 23 Vireo crassiroslr is Andros, Bahamas MRW 24 Vireo crassirosîris Abaco, Bahamas MRW 25 Vireo crassirostris Abaco, Bahamas MRW 26 Vireo crassiroslris Abaco, Bahamas MRW 27 Vireo crassirostris Abaco, Bahamas MRW 28 Vireo crassiroslris Paredon Grande Cay (Cuba) MRW 29 Vireo crassirostris Paredon Grande Cay (Cuba) MRW 30 Vireo crassirostr~s Cayman Brac, Cayman 1. MRW 33 Vireo crassirostris Cayman Brac, Cayman 1. MRW 34 Vireo crassirostris Grand Cayman, Cayman 1. MRW 40 Vireo crassirostris Providenciaies, Turks & Caicos 1%VO6 18 MRW 42
97 Specimens Used in Analysis - Cont'd Centre for Biodiversity, Royal Ontario Museum, Toronto, Ontario, Canada
Location Date of collection Collection #
Vite0 crassirmtris Providenciales, Turks & Caicos 19970619 MRW 43 Vireo crassirostris Providenciales, Turks & Caicos 1997061 9 MRW 44 Vireo crassirostris Providenciales, Turks & Caicos 1997061 9 MRW 45 Vireo crassirosfris Providenciales, Turks & Caicos 19970619 MRW 46 Vire0 crussirusfris ProvidenciaIes, Turks & Caicos 19970620 MRW 47 Vireo crassirostris Providenciales, Turks & Caicos 19970620 MRW 48 Vire0 crassirostris Providenciales, Turks & Caicos 19970620 MRW 49 Vireo crassirostris Providenciales, Turks & Caicos 19970620 MRW 50 Vireo crassirostrzs Providenciales, Turks & Caicos 19970620 MRw51 Vireo crussiruslris Middle Caicos, Turks & Caicos 19970622 MRW 55 Vireo crarsiroslris Middle Caicos, Turks & Caicos 19970622 MRW 56
Vireo griseus Texas, U. S.A. JCB 5660 Vireo griseus Texas, U.S.A. JCB 5661 Vireo griseus Texas, U.S.A. JCB 5669
VÏreo pallens Belize MKP1832 Vireo pallens Mexico JCB5727 Vireopaliens Belize JCB5728
Vireo nanus Dorninican RepubIic 19960421 MKP2689 Vireo nanus Dominican Republic 19960422 MKP2692 Vireo nanus Dominican Republ ic 19960422 MW2693
Vireo gundlachii Cuba (Matanzas) 19930804 JCB5722 Appendix 3
THICK-BILLED VIRlcO SEQUENCES &Strand)
1 1111111112 2222222223 3333333334 4444444445 5555555556 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 MRWOOlL ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW002L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW003L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRWOO4L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW005L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW006L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW007L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW008L ATGTTTTACA TAGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW009L ATGTTTTACA TAGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRWOlOL ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRWOllL ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRWO12L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW013L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW014L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW015L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRWO16L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW017L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW018L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW019L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRWO2OL ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW021L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW022L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW023L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW024L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW025L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW026L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW027L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW028L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW029L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW030L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW033L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW034L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW040L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW042L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW043L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRWO44L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW045L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW046L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW047L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW048L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW049L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW050L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW051L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW055L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG MRW056L ATGTTTTACA TGGGTCATTT AGGTATGTAT TACTTTGCAT ACAATTTATG TCCACATCAG Appendix 3 100 THICK-BILLED VIRE0 SEQUENCES (Lstrand) (Cont.)
MRWOOlL ACATTATATT AATGTAGGAT ATTCCACATA ACATGTMTG CTCGTCCTCA TTAAACTCAC MRW002L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW003L ACATTATATT AATGTAGGAT ATTCCACATA GTATGTAATG CTCGTCCTCA TTAAACTCAC MRW004L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW005L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW006L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW007L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWOOBL ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWOOSL ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWOlOL ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWOllL ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW012L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW013L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW014L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWO15L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW016L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWO17L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW018L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWO19L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWOZOL ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW021L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW022L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW023L ACATTATATT AATGTAGGAT ATTCCACATA ATATGTAATG CTCGTCCTCA TTAAACTCAC MRW024L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW025L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW026L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW027L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW028L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW029L ACATTATATT AiiTGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW030L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW033L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW034L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRWO4OL ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW042L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW043L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW044L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW045L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW046L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW047L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTRAACTCAC MRW048L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW049L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW050L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW051L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC MRW055L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAZLACTCAC MRW056L ACATTATATT AATGTAGGAT ATTCCACATA ACATGTAATG CTCGTCCTCA TTAAACTCAC Appendix 3 101 TMCK-BKLED VIRE0 SEQUENCES (L-SM)(Cent.)
1111111111 llllflllll 1111111111 1111111111 11111l,l111 llllllllll 2222222223 3333333334 4444444445 5555555556 6666666667 7777777778 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 MRWOOlL ATATTATAGC CCATATCAAT GCTAATCGGA CAGGTATATT GCTAGGCACA TTCCCATTTC MRWOOZL ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW003L ATATTATAGC CCATATCAAT GCTAATCGGA CAGGTATACT GCTAGGCACA TTCCCATTTC MRWOOIL ATATTATAGC CCATATCAAT GCTAATCGGA CAGGTGTACT ACTAGGCACA TTCCCATTTC MRW005L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW006L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW007L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW008L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW009L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRWOlOL ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRWOllL ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW012L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW013L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRWOl4L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW015L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRWO16L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW017L ATATTATAGC CCATATTAGT GTTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW018L ATATTATAGC CCATATTAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW019L ATATTATAGC CCATATCAAT GCTAATCGGA CAGGTATACT GCTAGGCACA TTCCCATTTC MRW020L ATATTATAGC CCATATCAAT GCTAATCGGA CAGGTATACT GCTAGGCACA TTCCCATTTC MRW02lL ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW022L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW023L ATATTATAGC CCATATCAAT GCTAATCGGA CAGGTACACT ACCAGGCACA TTCCCATTTC MRW024L ATATTATAGC CCATATCAAT GCTAATCGGA CAGGTATACT GCTAGGCACA TTCCCATTTC MRIO25L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW026L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW027L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTACACT ACTAGGCACA TTCCCATTTC MRW028L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW029L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW030L ATATTATAGC CCATATCAGT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW033L ATATCATAGC CCATATTAAT GCTaATCGGA CAGGTACACT ACTAGGCACA TTCCCATTTC MRW034L ATATCATAGC CCATATTAAT GCTAATCGGA CAGGTACACT ACTAGGCACA TTCCCATTTC MRW040L ATATTATAGC CCATATTAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW042L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW043L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW044L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW045L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW046L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW047L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW048L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW049L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW050L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW051L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW055L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC MRW056L ATATCATAGC CCATATCAAT GCTAATCGGA CAGGTATACT ACTAGGCACA TTCCCATTTC Appenâix 3 102 THICK-BILLED VlREO SEQUENCES (L-sb.and) (Cm)
ll1llfllll 1111111112 2222222222 2222222222 2222222222 2222222222 8888888889 9999999990 0000000001 1111111112 2222222223 3333333334 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 MRWOOlL AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWOOPL AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW003L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW004L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWOOSL AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW006L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW007L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW008L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW009L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW010L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWO11L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWOl2L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWO13L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWOl4L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW015L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWO16L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW017L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW018L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW019L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW020L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW021L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRWO22L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW023L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW024L AGGTACCATA AACCCAAATG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW025L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW026L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW027L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW028L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW029L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW030L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW033L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW034L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW040L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGATCGA MRW042L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW043L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW044L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRWO~SLAGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW046L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW047L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW048L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW049L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW050L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRWOSlL AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW055L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA MRW056L AGGTACCATA AACCCAAGTG ATCCTACCTC CCCCCAGGGA CAAGCGTCAC CCGAGGTCGA Appendix 3 103 THICK-BiLLED VIRE0 SEQüENCES (L-strand) (Cont.) 2222222222 2222222222 2222222222 2222222222 2222222222 2222222223 4444444445 5555555556 6666666667 7777777778 8888888889 9999999990 1234567090 1234567890 1234567890 1234567890 1234567890 1234567890 MRWOOlL GACTGTTTCC CTACGCCCAA CTCTATCTCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOOSL GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW003L GACTGTTTCC CTACGCCCAA CTTTATTCCT AGTATACGAG AGTTATCCTA GTACATAATT MRWOOIL GACTGTTTCC CTACGCCCAA CTCTACTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOOSL GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOO6L GACTGTTTCC CTACGCCCAA CTCTACTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW007L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW008L GACTGTTTCC CTACGCCCAA CTCCATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOO9L GACTGTTTCC CTACGCCCAA CTCCATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOlOL GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOllL GACTGTTTCC CTACGCCCAA CTCCATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW012L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW013L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOl4L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW015L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW016L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW017L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW018L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOl9L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOSOL GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW021L GACTGTTTCC CTACGCCCAA CTCTACTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW022Z GACTGTTTCC CTACGCCCAA CTCTACTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW023L GACTGTTTCC CTACGCCCAA CTCTATCTCT AGTATACGAG AGTTATCCTA GTACATAACT MRWO24L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW025L GACTGTTTCC CTACGCCCAA CTCTACTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW026L GACTGTTTCC CTACGCCCAA CTCTACTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW027L GACTGTTTCC CTACGCCCAA CTTTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW028L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWO29L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWO3OL GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW033L GACTGTTTCC CTACGCCCAA CTCTATCCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW034L GACTGTTTCC CTACGCCCAA CTCTATCCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWO4OL GACTGTTTCC CTACGCCCGA CTCTATCCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW042L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW043L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW044L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW045L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW046L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW047L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW048L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW049L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOSOL GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRWOSlL GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW055L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT MRW056L GACTGTTTCC CTACGCCCAA CTCTATTCCT AGTATACGAG AGTTATCCTA GTACATAACT Appendix 3 104 THICK-BILLED WREO SEQUENCES (L-strand) (Cont) 3333333333 3333333333 3333333333 3333333333 3333333333 3333333333 0000000001 1111111112 2222222223 3333333334 4444444445 5555555556 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 MRWOOlL GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWOO2L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW003L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW004L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW005L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW006L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW007L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW008L GAACCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW009L GAACCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWOlOL GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWOllL GAACCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWO12L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW013L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW014L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWO15L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAa TGTCCCTTTC CAACAGCTTT MRW016L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW017L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW018L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW019L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW020L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW021L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW022L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWO23L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW024L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW025L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW026L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW027L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW028L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW029L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW030L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CZLACAGCTTT MRW033L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW034L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW040L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW042L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW043L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW044L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW045L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW046L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW047L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW048L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW049L GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWOSOL GAACCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRWO5lL GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW055L GAaTCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT MRW056Z GAATCCTCTA GTCCATAAGT TTCGCCCACC TCCTAGGGAA TGTCCCTTTC CAACAGCTTT Appendix 3 105 THICK-BiLLED VIRE0 SEQUENCES (L-strand) (Cont.) 3333333333 3333333333 333333333 6666666667 7777777778 888888888 1234567890 1234567890 123456789 MRWOOIL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW002L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW003L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOOQL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOOSL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOOGL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW007L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOOBL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOOSL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOlOL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOllL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW012L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW013L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWOl4L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW015L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWO16L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW017L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW018L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW019L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW020L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW02I.L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW022L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW023L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW024L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW025L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWO 2 6L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW027L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWO28L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW029L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW030L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW033L CAAGTACTCA CAAGCCAGGG AGCCT??TT MRW034L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRWO4OL CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW042L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW043L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW044L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW045L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW046L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW047L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW048L CAAGTACTCA CAAGCCAGGG ????????? MRW049L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW050L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW051L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW055L CAAGTACTCA CAAGCCAGGG AGCCTGGTT MRW056L CAAGTACTCA CAAGCCAGGG AGCCTGGTT Appendix 4
VARiAi3LE SITES - THICK-BILLED ViREO SEQUENCES (L-strand)
1111111 1112222222 23 1992334555 6693566666 90 2125792679 1386934678 94 MRWOO2L GACTCGCATC ATGAACTTTC CT MRWOOlL .....A...T G...... CT .. MRW003L .GT..A.... G....T.... T. MRW004L .....A.G...... C.. .. MRW005L ...... MRW006L ...... C.. .. MRWDO7L ...... MRWOOBL A...... C... .C MRWOO9L A...... C... .C MRWO10L ...... MRWOllL ...... C... .C MRW012L ...... MRW013L ...... MRW014L ...... MRWO15L ...... MRWO16L ...... MRW017L ....T.T...... MRW018L ....T...... MRW019L .....A. ... G...... MRW020L .....A.... G...... MRWO21L ...... C.. .. MRW022L ...... C.. .. MRW023L ..T..A..C. .C.G....CT .. MRW024L .....A.... G.A...... MRW025L ...... C.. .. MRW026L ...... C.. .. MRW027L ...... C...... T...... MRW028L ...... MRW029L ...... MRW030L ...... MRW033L ...CTA..C...... C. .. MRW034L ...CTA..C...... C. .. MRW040L ....TA...... G...C. .. MRW042L ...C.A...... G...... MRW043L ...C.A...... G...-.. .. MRW044L .. .C.A...... G...... MRW045L ...C.A...... G...... MRW046L ...C.A...... G...... MRW047L ...C.A...... G...... MRW048L ...C.A...... G...... MRW049L ...C.A...... G...... MRW050L ...C.A...... G...... C MRW051L ... C.A..-. ...G...-.. .. MRW055L ...C.A...... G...... MRW056L ...C.A...... G...... Appendix 5 NUCLEOTIDE COMPOSITION OF THICK-BILLED VIRE0 SEQUENCES
Al1 values in per cent (8) except Totals A T C G Total MRWOOlL 28.3 29.3 26.5 15.9 389 MRWOOZL 28.3 29.0 26.7 15.9 389 MRW003L 28.0 29.8 26.0 16.2 389 MRW004L 28.3 28.8 27.0 15.9 389 MRW005L 28.3 29.0 26.7 15.9 389 MRWOO6L 28.3 28.8 27.0 15.9 389 MRWOO7L 28.3 29.0 26.7 15.9 389 MRWOO8L 28.5 28.5 27.2 15.7 389 MRWO09L 28.5 28.5 27.2 15.7 389 MRWOlOL 28.3 29.0 26.7 15.9 389 MRWOllL 28.3 28.5 27.2 15.9 389 MRW012L 28-3 29.0 26.7 15.9 389 MRW013L 28.3 29.0 26.7 15.9 389 MRW014L 28.3 29.0 26.7 15.9 389 MRWO15L 28.3 29.0 26.7 15.9 389 MRWO16L 28.3 29.0 26.7 15.9 389 MRW017L 28.3 29.6 26.2 15.9 389 MRW018L 28.3 29.3 26.5 15.9 389 MRW019L 28.3 29.0 26.7 15.9 389 MRWO2OL 28.3 29.0 26.7 15.9 389 MRWO21L 28.3 28.8 27.0 15.9 389 MRWO22L 28.3 28.8 27.0 15.9 389 MRW023L 28.3 28.8 27.0 15.9 389 MRW024L 28.5 29.0 26.7 15.7 389 MRW025L 28.3 28.8 27.0 15.9 389 MRWO26L 28.3 28.8 27.0 15.9 389 MRW0271; 28.3 29.0 26.7 15.9 389 MRWO28L 28.3 29.0 26-7 15.9 389 MRWO29L 28.3 29.0 26.7 15-9 389 MRW03OL 28.3 29.0 26.7 15.9 389 MRW033L 28.7 28.7 27.4 15.2 387 MRWO34L 28.5 28.5 27.2 15.7 389 MRWO40L 28.3 29.0 26.7 15.9 389 MRW042L 28.3 28.8 27-0 15.9 389 MRWO43L 28.3 28.8 27.0 15.9 389 MRW044L 28.3 28.8 27.0 15.9 389 MRW045L 28.3 28.8 27.0 15.9 389 MRW046L 28.3 28.8 27.0 15.9 389 MRW047L 28.3 28.8 27.0 15.9 389 MRWO48L 28.7 28.7 27.1 15.5 380 MRW049L 28.3 28.8 27.0 15.9 389 MRWOSOL 28.3 28.5 27.2 15.9 389 MRWO51L 28.3 28.8 27.0 15.9 389 MRW055L 28.3 28.8 27.0 15.9 389 MRW056L 28.3 28.8 27. O 15.9 389 Total 28.3 28.9 26.9 15.9 16716 Appendix 6
HURRICANE DATA Permission to access data provided by Dr. Robert Sheets, director of National Hurricane Center, Miami, Florida and Mr. Neal Loft, National Climatic Data Center, Ashville, North Carolina.
This is a list of Atlantic hurricanes since 1886. Provided are charts on the track of the storrn plus a text based table of tracking information. The table includes position in latitude and longitude, maximum sustained winds in knots, and central pressure in millibars. The chart codes intensity 1-5 (category based on Saffir-Simpson scale):
Type Category Pressure Winds Surge mb knts mph ft Depression TD ----- < 34 < 39 ----- Tropical Storm TS ----- 34- 63 39- 73 ----- Hurricane 1 > 980 64- 82 74- 95 4- 5 Nurricane 2 965-980 83- 95 96-110 6- 8 Hurricane 3 945-965 96-112 111-130 9-12 iiurricane 4 920-945 113-134 131-155 13-18 Hurricane 5 < 920 > 134 > 155 > 18
HURRICANE DATA - BAHAMAS
Date: 25 AUG-8 SEP 1979 Hurricane DAVID ADV LAT LON TIME WIND PR STAT TROPICAL DEPRESSION TROPICAL DEPRESSION TROPICAL DEPREXS ION TROPICAL STORM TROPICAL STORM TROPICAL STORM TROPICAL STORM HURRICANE-1 HURRICANE-1 HURRICANE-2 HURFUCANE-4 HURRICANE-4 HURRICAETE-4 HURRICANE- 4 HURRICANE-4 HURRICANE-4 HURRICANE-4 HURRICANE-4 HURRICANE-4 HURRICANE- 5 HURRICANE-5 HURRICANE-5 HURRICANE-5 =RICANE-5 Appendix 6 109
HURRICANE DATA - BAHAMAS - (cont.) Date: 25 AuG-8 SEP 1979 Hurricane DAVID ADV LAT LON TIME WIND PR STAT
145 927 HURRICANE-5 150 926 HURRICANE-5 130 953 HURRICANE-4 100 978 HURRICANE-3 65 1002 HURRICANE-1 60 1002 TROPICAL STORM 65 997 HURRICANE-1 70 990 HURRICANE-1 70 984 HURRICANE-1 75 979 HURRICANE-1 80 976 HURRICANE-1 80 974 HURRICANE-1 85 973 HURRICANE-2 85 972 HURRICANE-2 85 971 HURRICANE-2 85 970 HURRICANE-2 85 970 HURRICANE-2 80 970 HURRICANE-1 65 972 HURRICANE-1 55 976 TROPICAL STORM 45 980 TROPICAL STORM 40 984 TROPICAL STORM 40 987 TROPICAL STORM 40 989 TROPICAL STORM 40 991 TROPICAL STORM 40 992 EXTRATROPICAL STORM 45 991 EXTRATROPICAL STORM 50 988 EXTRATROPICAL STORM 50 987 EXTRATROPICAL STORM 55 986 EXTRATROPICAL STORM 60 985 EXTR74TROPICAL STORM
Date: 15-23 NOV 1985 Hurricane KATE ADV LAT LON TIME WIND PR STAT 1 21.10 -63.80 11/15/182 35 999 TROPICAL STORM 2 21.60 -63.90 11/16/002 45 998 TROPICAL STORM 3 21.70 -64.20 11/16/062 50 996 TROPICAL STORM 4 21.50 -64.80 11/16/12Z 55 993 TROPICAL STORM 5 21.10 -65.30 11/16/182 70 987 HURRICANE-1 6 20.70 -66.00 11/17/002 75 981 HURRICANE-1 7 20.40 -66.40 11/17/062 75 984 HURRICME-1 8 20.70 -67.30 11/17/122 75 982 HURRICANE-1 9 21.10 -68.80 11/17/182 80 977 KURRICANE-1 10 21.40 -70.00 11/18/00Z 80 976 HURRICANE-1 11 21.60 -71.80 11/18/062 80 975 HURRICANE-1 12 21.60 -73.30 11/18/122 80 975 HURRICANE-1 13 21.90 -75.10 11/18/182 85 972 HURRICANE-2 14 22.10 -76.80 11/19/002 95 967 HURRICANE-2 15 22.10 -78.40 11/19/062 95 968 HURRICANE-2 Appendix 6 1 10 HURRICANE DATA - BAHAMAS - (cont.) Date: 15-23 NOV 1985 Hurricane KATE ADV LAT LON T IME WIND PR STAT
90 971 HURRICANE-2 80 976HURRICANE-1 85 972 HURRICANE-2 95 968 HURRICANE-2 105 956 HURRICANE-3 105 955 HURRICANE-3 105 954 HURRICANE-3 100 961 HURRICANE-3 95 965 HURRICANE-2 85 967 HURRICANE-2 80 975 HURRICME-I 65 983 HURRICANE-1 50 990 TROPICAL STORM 45 996 TROPICAL STORM 40 1003 TROPICAL STORM 35 1005 TROPICAL STORM 35 1006 TROPICAL STORM 35 1006 EXTRATROPICAL STORM
Date: 20-26 SEP 1987 Hurricane EMILY ADV LAT LON TIm WIND PR STAT 1 9.80 -51.30 09/20/00Z 25 1008 TROPICAL DEPRESSION 2 10.40 -53.00 09/20/06Z 25 1007 TROPICAL DEPRESSION 3 10.90 -54.70 09/20/12Z 30 1006 TROPICAL DEPRESSION 4 11.40 -56.40 09/20/18Z 35 1005 TROPICAL STORM 5 12.00 -58.00 09/21/002 40 1004 TROPICAL STORM 6 12.40 -59.70 09/21/06Z 40 1004 TROPICAL STORM 7 13.10 -61.30 09/21/12Z 45 1004 TROPICAL STORM 8 13.70 -63.10 09/21/18Z 50 1002 TROPICAL STORM 9 14.50 -64.70 09/22/00Z 60 992 TROPICAL STORM 10 15.10 -66.30 09/22/062 80 978 HURRICANE-1 11 15.90 -67.70 09/22/122 90 971 HUMICANE-2 12 16.70 -69.10 09/22/182 110 958 HURRICANE-3 13 17.80 -70.40 09/23/002 105 960 HURRICANE-3 14 19.00 -71.50 09/23/06Z 70 984 HURRICANE-1 15 20.00 -72.30 09/23/12Z 55 1004 TROPICAL STORM 16 20.90 -72.80 09/23/18Z 40 1003 TROPICAL STORM 17 22.00 -73.00 09/24/00Z 40 1000 TROPICAL STORM 18 23.20 -73.00 09/24/062 45 1001 TROPICAL STORM 19 24.40 -72.70 09/24/122 45 1002 TROPICAL STORM 20 26.00 -72.00 09/24/182 45 1002 TROPICAL STORM 21 28.00 -70.50 09/25/002 45 999 TROPICAL STORM 22 30.20 -68.00 09/25/062 70 985 HURRICANE-1 23 32.40 -64.60 09/25/12Z 80 974 HURRICANE-1 24 35.00 -60.00 09/25/182 80 974 HURRICANE-1 25 38.00 -55.00 09/26/00Z 75 976 HURRICANE-1 26 41.20 -49.00 09/26/06Z 70 979 HURRICANE-1 27 44.80 -42.50 09/26/122 65 983 HURRICANE-1 28 49.00 -36.00 09/26/18Z 55 994 EXTRATROPICAL STORM Appendix 6 11 1
HURFtICANE DATA - BA- - (cont.) Date: 20-26 SEP 1987 Hurricane EMILY ADV LAT LON T IME WIND PR S TAT 1 9.80 -51.30 09/20/002 25 1008 TROPICAL DEPRESSION 2 10.40 -53.00 09/20/062 25 1007 TROPICAL DEPRESS ION 3 10.90 -54.70 09/20/12Z 30 1006 TROPICAL DEPRESSION 4 11.40 -56.40 09/20/18Z 35 1005 TROPICAL STORM 5 12.00 -58.00 09/21/00Z 40 1004 TROPICAL STORM 6 12.40 -59.70 09/21/062 40 1004 TROPICAL STORM 7 13.10 -61.30 09/21/12Z 45 1004 TROPICAL STORM 8 13.70 -63.10 09/21/18Z 50 1002 TROPICAL STORM 9 14.50 -64.70 09/22/002 60 992 TROPICAL STORM 10 15.10 -66.30 09/22/062 80 978 HURRICANE-1 11 15.90 -67.70 09/22/12Z 90 971 HURRICANE-2 12 16.70 -69.10 09/22/18Z 110 958 HURRICNNE-3 13 17.80 -70.40 09/23/002 105 960 HURRICYW3-3 14 19.00 -71.50 09/23/062 70 984 HURRICANE-1 15 20.00 -72.30 09/23/12Z 55 1004 TROPICAL STORM 16 20.90 -72.80 09/23/182 40 1003 TROPICAL STORM 17 22.00 -73.00 09/24/00Z 40 1000 TROPICAL STORM 18 23.20 -73.00 09/24/062 45 1001 TROPICAL STORM 19 24.40 -72.70 09/24/122 45 1002 TROPICAL STORM 20 26.00 -72.00 09/24/182 45 1002 TROPICAL STORM 21 28.00 -70.50 09/25/00Z 45 999 TROPICAL STORM 22 30.20 -68.00 09/25/062 70 985 HURRI CANE - 1 23 32.40 -64.60 09/25/12Z 80 974 HURRICANE-1 24 35.00 -60.00 09/25/182 80 974 HURRICANE-1 25 38.00 -55.00 09/26/002 75 976 HURRICANE-1 26 41.20 -49.00 09/26/062 70 979 HURRICANE-1 27 44.80 -42.50 09/26/122 65 983 HURRICANE-1 28 49.00 -36.00 09/26/18Z 55 994 EXTRATROPICAL STORM
Date: 31 JUL-6 AUG 1995 Hurricane ERIN ADV LAT LON TIME WIND PR S TAT 45 1004 TROPICAL STORM 50 1003 TROPICAL STORM 55 999 TROPICAL STORM 60 997 TROPICAL STORM 70 992 HURRICANE-1 75 988 HURRICAEJE-1 75 985 HURRICANE- 1 75 980 HURRICANJ3- 1 75 982 HURRICANE-1 75 985 HURRICANE-1 50 990 TROPICAL STORM 60 988 TROPICAL STORM 65 985 HURRICANE-1 70 979 HURRICANE-1 80 974 HURRICANE-1 65 985 HURRICANE-1 45 997 TROPICAL STORM 35 1001 TROPICAL STORM 20 1003 TROPICAL DEPRESSION 20 1003 TROPICAL DEPRFSSION Appendix 6 1 12
HURRICANE DATA - BAHAMAS - (cont. ) Date: 31 JUL-6 AUG 1995 Hurricane ERIN ADV LAT LON TIME WIND PR STAT 21 34.80 -90.20 08/05/002 20 1003 TROPICAL DEPRESSION 22 35.40 -90.10 08/05/062 20 1003 TROPICAL DEPRESSION 23 36.30 -89.80 08/05/122 20 1003 TROPICAL DEPRESSION 24 37.50 -88.80 08/05/182 20 1003 TROPICAL DEPRESSION 25 38.40 -86.80 08/06/00Z 20 1003 TROPICAL DEPRESSION 26 38.70 -84.90 08/06/06Z 20 1005 TROPICAL DEPRESSION 27 38.80 -82.00 08/06/122 20 1008 TROPICAZ, DEPRESSION
Date: 28 AUG-12 SEP 1995 Hurricane LUIS ADV LAT LON TIME WIND PR STAT 1008 TROPICAL DEPRESSION 1005 TROPICAL STORM LOO0 TROPICAL STORM 1000 TROPICAL STORM 1003 TROPICAL STORM 1005 TROPICAL STORM 1005 TROPICAL STORM 1005 TROPICAL STORM 1002 TROPICAL STORM 998 TROPICAL STORM 992,HUMICANE-1 979 HURRICANE-1 971 HURRICANE-2 965 HURRICANE-3 958 HURRICANE-3 950 HURRICANE-4 948 HURRICANE-4 948 HURRICANE-4 948 HURRICANE-4 948 HURRICANE-4 948 HURRICANE-4 948 HURRICANE-4 948 HURRICANE-4 948 HURRICAIW-4 945 HURRICANE-4 942 HURRICANE-4 940 HURRICANE-4 945 HURRICANE-4 943 HURRICANE-4 940 HURRICANE-4 939 HURRICANE-4 945 HURRICANE-4 944 HURRICANEI-4 942 HURRICANE-4 939 HURRICANE-4 943 HURRICANE-4 940 HüRRICANE-4 938 HURRICANE-4 936 HURRICANE-4 941 WURRICANE-4 938 HURRICANE-4 935 HURRICm-4 Appendix 6 1 13
HURRICANE DATA - BAHAMAS - ( cont .) Date: 28 AUG-12 SEP 1995 Hurricane LUIS ADV LAT LON T IME WIND PR STAT
939 HURRICANE-4 941 HURRICANE-3 944 HURRICANE-3 945 HURRICANE-3 949 HURRICANE-2 90 952 HURRICANE-2 85 955 HURRICANE-2 85 959 HURRICANE-2 85 963 HURRIC31NE-2 85 961 HURRICANE-2 85 966 EXTRATROPICAL STORM-1 95 965 EXTRATROPICAL STORM-1 105 963 EXTRATROPICAL STORM-1 90 960 EXTRATROPICAL STORM-1 75 958 EXTRATROPIW STORM-1 60 955 EXTRATROPICAL STORM 60 950 EXTRATROPICAL STORM 60 955 EXTRATROPICAL STORM 50 960 EXTRATROPICAL STORM
Date: 16-28 AUG 1992 Hurricane ANDREW ADV LAT LON TIME WIND PR STAT 1 10.80 -35.50 08/16/182 1010 TROPICAL DEPRESSION 2 11.20 -37.40 08/17/00Z 1009 TROPICAL DEPRESSION 3 11.70 -39.60 08/17/062 1008 TROPICAL DEPRESSION 4 12.30 -42.00 08/17/122 1006 TROPICAL STORM 5 13.10 -44.20 08/17/182 1003 TROPICAL STORM 6 13.60 -46.20 08/18/002 1002 TROPICAL STORM 7 14.10 -48.00 08/18/062 1001 TROPICAL STORM 8 14.60 -49.90 08/18/122 1000 TROPICAL STORM 9 15.40 -51.80 08/18/182 1000 TROPICAL STORM 10 16.30 -53.50 08/19/002 1001 TROPICAL STORM 11 17.20 -55.30 08/19/062 1002 TROPICAL STORM 12 18.00 -56.90 08/19/122 1005 TROPICAL STORM 13 18.80 -58.30 08/19/182 1007 TROPICAL STORM 14 19.80 -59.30 08/20/00Z 1011 TROPICAL STORM 15 20.70 -60.00 08/20/062 1013 TROPICAL STORM 16 21.70 -60-70 08/20/122 1015 TROPICAL STORM 17 22.50 -61.50 08/20/182 1014 TROPICAL STORM 18 23.20 -62.40 08/21/00Z 1014 TROPICAL STORM 19 23.90 -63.30 08/21/06Z 1010 TROPICAL STORM 20 24.40 -64-20 08/21/12Z 1007 TROPICAL STORM 21 24.80 -64.90 08/21/182 1004 TROPICAL STORM 22 25.30 -65.90 08/22/002 1000 TROPICAL STORM 23 25.60 -67.00 08/22/062 994 TROPICAL STORM 24 25.80 -68.30 08/22/122 981 HURRICANE-1 25 25.70 -69.70 08/22/18Z 969 HURRICANE-1 26 25.60 -71.10 08/23/002 961 HURRICRNE-2 27 25.50 -72.50 08/23/062 947 HURRICANE-3 28 25.40 -74.20 08/23/122 933 HURRICANE-4 Appendix 6 1 14 HURRICANE DATA - BAHAMAS - Icont.) Date: 16-28 AUG 1992 Hurricane ANDREW ADV LAT LON TIME WIND PR STAT
125 930 HURRIW-4 120 937 WURRICANE-4 110 951 HWRRICANE-3 115 947 HURRICANE-4 115 943 HURRICANE-4 115 948 HURRICANE-4 115 946 HURRICANE-4 120 941 HURRICANE-4 120 937 HURRICANE-4 115 955 HURRICANE-4 80 973 HURRICANE-1 50 991 TROPICAL STORM 35 995 TROPICAL STORM 30 997 TROPICAL DEPRESSION 30 998 TROPICAL DEPRESSION 25 999 TROPICAL DEPRESSION 20 1000 TROPICAL DEPRESSION 20 1000 TROPICAL DEPRESSION
Date: 5-17 JLTL 1996 Hurricane BERTHA ADV LAT LON TIME WIND PR STAT 1 9-80 -34 .O0 07/05/00Z 1009 TROPICAL DEPRESSION 2 10.20 -36.30 07/05/062 1008 TROPICAL DEPRESSION 3 11.00 -39.00 07/05/122 1007 TROPICAL STORM 4 12.00 -41.20 07/05/182 1006 TROPICAL STORM 5 12.70 -43.90 07/06/00Z 1005 TROPICAL STORM 6 13.10 -46.60 07/06/062 1004 TROPICAL STORM 7 13.70 -48.70 07/06/122 1002 TROPICAL STORM 8 14.20 -51.00 07/06/18Z 1000 TROPICAL STORM 9 14.90 -52.90 07/07/00Z 999 TROPICAL STORM 10 15.60 -54-80 07/07/062 997 TROPICAL STORM 11 16.40 -56.90 07/07/122 995 TROPICAL STORM 12 16.50 -58.40 07/07/182 992 HURRICANE-1 13 17.00 -60.10 07/08/002 988 HURRICANE-1 14 17.50 -61-80 07/08/06Z 985 HURRICANE-1 15 18.00 -63.50 07/08/12Z 983 HURRICANE-1 16 18.60 -64.90 07/08/18Z 978 HURRICANE-1 17 19.40 -66.10 07/09/002 970 HURRICANE-1 18 20.30 -67.70 07/09/06Z 960 HURRICANE-3 19 21.40 -69.40 07/09/122 965 HURRICANE-3 20 22.50 -71.10 07/09/18Z 967 HURRICANE-2 21 23.60 -72.60 07/10/00Z 969 HURRICANE-2 22 24.50 -74.00 07/10/06Z 971 HURRICANE-1 23 25.40 -75.30 07/10/122 968 HURRICANE-1 24 26.40 -75.80 07/10/18Z 966 HURRICANE-1 25 27.50 -76.40 07/11/00Z 968 HURRICANE-1 26 28.30 -76.80 07/11/062 972 HURRICANE-1 27 29.20 -77.50 07/11/12Z 977 HURRICANE-1 28 30.00 -78.00 07/11/182 980 HUREtICANE-1 29 30.70 -78.30 07/12/00Z 982 HURRICANE-1 30 31.20 -78.60 07/12/062 984 HURRICANE-1 975 HURRICANE-2 Appendk 6 1 15
HURRICANE DATA - B-S - ( cont .) Date: 5-17 JUL 1996 Hurricane BERTHA ADV LAT LON TIME WIND PR STAT
90 974 HURRICANE-2 65 993 HURRICANE-1 60 993 TROPICAL STORM 60 994 TROPICAL STORM 60 994 TROPICAL STORM 60 994 TROPICAL STORM 55 995 TROPICAL STORM 50 995 EXTRATROPICAL STORM 50 995 EXTRATROPICAL STORM 50 995 EXTRATROPICAL STORM 45 996 EXTRATROPILAL STORM 40 996 EXTRATROPICAL STORM 40 996 EXTRATROPICAL STOW 40 991 EXTRATROPICAL STORM 40 988 EXTRATROPICAL STORM 45 988 EXTRATROPICAL STORM 45 985 EXTRATROPICAL STORM 40 993 EXTRATROPICAL STORM 35 1001 EXTRATROPICAL STORM Date: 3-16 SEP 1996 Hurricane HORTENSE ADV LAT LON T IME WIND PR STAT 1 14.90 -41.00 09/03/122 25 1006 TROPICAL DEPRESSION 2 14.90 -42.70 09/03/18Z 30 1006 TROPICAL DEPRESSION 3 14.80 -44.10 09/04/002 30 1006 TROPICAL DEPRESSION 4 14.70 -45.40 09/04/062 30 1006 TROPICAL DEPRESSION 5 14.60 -46.60 09/04/12Z 30 1006 TROPICAL DEPRESSION 6 14.70 -47.50 09/04/18Z 30 1006 TROPICAL DEPRESSION 7 14.90 -48.40 09/05/002 30 1006 TROPICAL DEPNSSION 8 14.80 -49.50 09/05/06Z 30 1006 TROPICAL DEPRESSION 9 14.50 -51-10 09/05/122 30 1006 TROPICAL DEPRESSION 10 14.30 -52-60 09/05/182 30 1006 TROPICAL DEPRESSION 11 14.40 -53.60 09/06/00Z 30 1006 TROPICAL DEPRESSION 12 14.60 -54.30 09/06/062 30 1006 TROPICAL DEPRESSION 13 14.70 -55.10 09/06/12Z 30 1006 TROPICAL DEPRESSION 14 14.90 -55.70 09/06/18Z 30 1006 TROPICAL DEPRESSION 15 15.20 -57.00 09/07/00Z 30 1006 TROPICAL DEPRESSION 16 15.40 -58.30 09/07/06Z 35 1005 TROPICAL STORM 17 15.60 -59.60 09/07/12Z 40 1004 TROPICAL STORM 18 15.80 -60.40 09/07/182 40 1000 TROPICAL STORM 19 16.10 -61.20 09/08/00Z 50 996 TROPICAL, STORM 20 16.10 -62.00 09/08/06Z 55 996 TROPICAL STORM 21 16.10 -62.80 09/08/122 60 996 TROPICAL STORM 22 16.10 -63.60 09/08/18Z 60 991 TROPICAL STORM 23 16.10 -64.10 09/09/002 60 990 TROPICAL STORM 24 10.10 -64.50 09/09/062 70 987 HURRICANE-1 25 16.30 -65.00 09/09/12Z 70 985 HURRICANE-1 26 16.60 -65.60 09/09/182 70 990 HURRICANE-1 27 17.10 -66.10 09/10/002 70 989 HURRICANE-1 28 18.00 -66.90 09/10/062 70 989 HURRICANE-1 29 18.30 -67.80 09/10/12Z 65 989 HTRRICANE-1 30 18.90 -68.40 09/10/182 65 990 HURRICANE-1 Appendix 6 1 16
HURRICANE DATA - BAHAMAS - (cent .) Date: 3-16 SEP 1996 Hurricane HORTENSE ADV LAT LON TIME WIND PR STAT 70 982 HURRICANE-1 75 975 HURRICANE-1 90 971 HURRICANE-2 95 970 HURRICANE-2 100 967 HURRICANE-3 105 962 HURRICANE-3 115 959 HURRICANE-4 115 946 HURRICANE-4 120 935 HURRICANE-4 115 942 HURRICANE-4 100 948 HURRICANE-3 100 948 HURRICANE-3 90 948 HURRICANE-2 90 955 HURRICANE-2 85 960 HURRICANE-2 75 960 HURRICANE-1 70 970 HURRICANE-1 65 980 HURRICANE-1 60 982 TROPICAL STORM 40 996 EXTRATROPICAL STORM 40 998 EXTRATROPICAL STORM 35 999 EXTRATROPICAL STORM
Date: 14-29 OCT 1996 Hurricane LILI ADV LAT LON TIME WIND PR STAT 1 12.80 -80.40 10/14/12Z 1006 TROPICAL DEPRESSION 2 13.40 -80.90 10/14/182 1005 TROPICAL DEPRESSION 3 14.10 -81.40 10/15/002 1005 TROPICAL DEPRESSION 4 14.80 -81.90 10/15/062 1005 TROPICAL DEPRESSION 5 15.40 -82.50 10/15/122 1004 TROPICAL DEPRESSION 6 16.10 -83.10 10/15/182 1003 TROPICAL DEPRESSION 7 16.80 -83.50 10/16/002 999 TROPICAL DEPRESSION 8 17.50 -83.80 10/16/062 998 TROPICAL STORM 9 18.20 -83.80 10/16/122 998 TROPICAL STORM 10 18.30 -84.50 10/16/18Z 996 TROPICAL STORM 11 18.20 -84.20 10/17/002 992 TROPICAL STORM 12 18.80 -83.70 10/17/06Z 990 TROPICAL STORM 13 19.60 -83.50 10/17/12Z 987 HURRICANE-1 14 20.50 -83.10 10/17/18Z 984 HURRICANE-1 15 21.30 -82.80 10/18/002 982 HURRICANE-1 16 21.80 -82.20 10/18/062 980 HURRICANE-1 17 22.40 -81.50 10/18/12Z 975 HURRICANE-2 18 22.50 -80.00 10/18/18Z 975 HuRR1CA.m-1 19 23.00 -78.20 10/19/00Z 975 HURRICANE-2 20 23.50 -76.20 10/19/062 970 HURRICANE-2 21 24.40 -74.00 10/19/12Z 960 HURRICm-3 22 25.50 -71.50 10/19/182 962 HURRICANE-2 23 26.90 -69.00 10/20/002 964 HURRZCANE-1 24 28.30 -67.00 10/20/062 968 HURRICANE-1 25 29.60 -65.00 10/20/122 960 HURRICANE-2 970 HURRICANE-1 Appendi 6 117 HURRICANE DATA - BAHAMAS - (cont.) Date: 14-29 OCT 1996 Hurricane LILI ADV LAT LON T IME WIND PR STAT 75 980 HURRICANE-1 70 985 HURRICANE-1 65 986 HURRICANE-1 65 987 HURRICANE-1 6 5 987 HURIIICANE-1 65 987 HURRICANE-1 65 987 HURRICANE-1 65 987 HURRICANE-1 65 987 HURRICANE-1 65 985 HURRICANE-1 65 981 HURRICANE-1 70 979 HURRICANE-1 70 979 HURRICAtTE-1 70 979 HURRICANE-1 70 979 HURRICANE-1 75 979 HURRICANE-1 80 977 HURRTCANE-1 80 973 HURRICANE-1 85 970 HURRICANE-2 80 971 HURRICANE-1 75 975 HU'RRICANE-I 70 978 HURRICANE-1 65 979 HURRICANE-1 60 980 TROPICaL STORM 55 978 TROPICAL STORM 55 980 EXTRATROPICAL STORM 55 978 EXTRATROPICAL STORM 55 973 EXTRATROPICAL STORM 55 973 EXTRATROPICAL STORM 55 973 EXTFUITROPICAL STORM 55 970 EXTRATROPICAL STORM 55 970 EXTRATROPICAL STORM Appendix 6 1 18 HURRICANE DATA - CAYMAN ISLANDS Date: 12-23 OCT 1944 Hurricane #11 ADV LAT LON TIME WIND PR STAT 1 15.00 -80.30 10/12/18Z 35 - TROPICAL STORM 2 16.10 -80.80 10/13/002 65 - HURRICANE-1 3 16.80 -80.90 10/13/06Z 65 - HURRICANE-1 4 17.40 -80.90 10/13/12Z 70 - HURRICANE-1 5 17.70 -80.90 10/13/182 70 - HURRICANE-1 6 17.90 -80.80 10/14/00Z 70 - HURRICANE-1 7 18.20 -80.70 10/14/06Z 70 - HURRICANE-1 8 18.50 -80.60 10/14/12Z 75 - HURRICANE-1 9 18.80 -80.60 10/14/18Z 75 - HURRICANE-1 10 19.00 -80.50 10/15/00Z 75 - HURRICANE-1 11 19.20 -80.50 10/15/062 75 - HURRICANE-1 12 19.30 -80.80 10/15/12Z 75 - HURRICANE-1 13 19.20 -81.30 10/15/182 80 - HURRICANE-1 14 19.20 -81.70 10/16/00Z 80 - HURRICANE-1 15 19.30 -82.10 10/16/062 80 - HURRICANE-1 16 19.40 -82.40 10/16/12Z 85 - HURRICANE-2 17 19.60 -82.70 10/16/182 85 - HURRICANE-2 18 19.90 -82.90 10/17/00Z 90 - HURRICANE-2 19 20.20 -82.90 10/17/062 95 - HURRICANE-2 20 20.60 -82.90 10/17/122 95 - HURRICANE-2 21 21.20 -82.90 10/17/18Z 100 - HURRICANE-3 22 21.90 -82.90 10/18/00Z 105 - HURRICANE-3 23 22.50 -82.90 10/18/06Z 1O5 - HURRICANE-3 24 23.10 -83.00 10/18/122 100 - HURRICANE-3 25 24.00 -82.90 10/18/182 105 - HURRICANE-3 26 25.30 -82.70 10/19/00Z 105 - HURRICANE-3 27 26.80 -82.40 10/19/06Z 65 - HURRICAIE-1 28 28.40 -82-10 10/19/12Z 65 968 HUR1IICANE-1 29 29.80 -81.70 10/19/182 60 978 TROPICAL STORM 30 31.20 -81.20 10/20/00Z 50 983 TROPICAL STORM 31 32.30 -80.80 10/20/06Z 45 987 TROPICAL STORM 32 33.50 -80.10 10/20/122 40 992 TROPICAL STORM 33 35.20 -78-50 10/20/182 35 996 TROPICAL STORM 34 36.90 -76.60 10/21/00Z 35 998 EXTRATROPICAL STORM 35 38.10 -75.00 10/21/06Z 40 997 EXTRATROPICAL STORM 36 39.40 -73.30 10/21/122 45 - EXTRATROPICAL STORM 37 41.10 -70.80 10/21/18Z 45 - EXTRATROPICAL STORM 38 42.90 -67.40 10/22/00Z 45 - EXTRATROPICAL STORM 39 44.90 -63.70 10/22/062 45 - EXTRATROPICAL STORM 40 47.00 -60.20 10/22/122 40 - EXTRATROPICAL STORM 41 49.20 -57.10 10/22/182 40 - EXTRATROPICAL STORM 42 52.20 -54.20 10/23/00Z 40 - EXTRATROPICAL STORM 43 56.10 -51.50 10/23/062 35 - EXTRATROPICAL STORM 44 60.00 -48.80 10/23/122 35 - EXTRATROPICAL STORM Appendix 6 119 HURRICANE DATA - CAYMAN ISLANDS (Cont.) Date: 8-20 SEP 1988 Hurricane GILBERT ADV LAT LON TIME WIND PR STAT 1008 TROPICAL DEPRESSION 1007 TROPICAL DEPRESSION 1006 TROPICAL DEPRESSION 1005 TROPICAL DEPRESSION 1004 TROPICAL STORM 1002 TROPICAL STORM 998 TROPICAL STORM 995 TROPICAL STORM 992 TROPICAL STORM 989 HURRICANE-1 982 HURRICANE-1 975 HURRICANE-2 970 HURRICANL-3 964 HURRICANE-3 962 HURRICANE-3 960 HURRICANE-3 960 HURRICm-3 960 HURRICANF,-3 952 HURRICANE-4 934 HUMICANE-4 905 HURRICANE-5 888 HURRICANE-5 889 HURRICANE-5 892 HURRICANE-5 925 HURRICANE-4 944 HURRICANE-3 949 HURRICANE-2 950 HURRICANE-2 950 HURRICANE-2 949 HURRICANE-3 946 HURRICANE-3 948 HURRICANE-4 950 HURRICANE-4 964 HURRICANE-1 988 TROPICAL, STORM 996 TROPICAL STORM 1000 TROPICAL DEPRESSION 1002 TROPICAL DEPRESSION 1004 TROPICAL DEPRESSION 1003 TROPICAL DEPRESSION 1003 TROPICAL DEPRESSION 1002 TROPICAL DEPRESSION 1001 TROPICAL DEPRESSION 999 TROPICAL DEPRESSION 998 EXTRATROPICAL, DEPRESSION 46 43.40 -86.50 09/20/002 25 995 EXTRATROPICAL DEPRESSION