Phylogeny and Phylogeography of Porites & Siderastrea (Scleractinia: Cnidaria) Species in The Caribbean and Eastern Pacific; Based on The Nuclear Ribosomal ITS Region --------------------------------------------- A Dissertation Presented to The Faculty of the Department of Biology and Biochemistry University of Houston --------------------------------------------- In Partial Fulfillment Of the Requirements for the Degree Doctor of Philosophy --------------------------------------------- by Zac Forsman May 2003
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Phylogeny and Phylogeography of Porites & Siderastrea (Scleractinia: Cnidaria) Species in The Caribbean and Eastern Pacific; Based on The Nuclear Ribosomal ITS Region
--------------------------------------------- A Dissertation
Presented to
The Faculty of the Department of Biology and Biochemistry
University of Houston
---------------------------------------------
In Partial Fulfillment
Of the Requirements for the Degree
Doctor of Philosophy
---------------------------------------------
by
Zac Forsman
May 2003
ii
Phylogeny and Phylogeography of Porites & Siderastrea (Scleractinia: Cnidaria) Species in The Caribbean and Eastern Pacific; Based on The Nuclear Ribosomal ITS Region
____________________________________ Zac H. Forsman
APPROVED:
____________________________________ Dr. Gerard M. Wellington, Chairman
____________________________________ Dr. George E. Fox, Co-Chairman
____________________________________ Dr. Michael Travisano
____________________________________ Dr. Stuart Hall
____________________________________ Dr. Ove Hoegh-Guldberg
____________________________________ Dean, College of Natural Sciences and Mathematics
iii
ACKNOWLEDGEMENTS
This work would not have been possible if it were not for an enormous network of
people, who have helped me in so many ways. I would like to thank my lovely wife Li
Zhang Forsman, for all of her love and support. I would like to thank my advisors Dr.
G. Wellington and Dr. G. Fox for their support and commitment of time and resources.
The following people have contributed samples from far across the globe without asking
anything in return: G. Wellington, M. Takabayashi, E. Neves, R. Johnsson, C. Guevara,
T. Snell, B. Victor, J. Mate, H. Guzman, and A. Fajardo. The following people have
provided technical or lab support: M. Larios-Sanz, U. Nagaswamy, B. Mulder, S. Posey,
S. Hardin, M. Travisano, D. Martinez, and D. Wells. Two undergraduate students
contributed numerous hours of lab work to this project; I would like to thank N.
Nnebuihe, and A. Konshack for their valuable contributions. I have received advice,
insight and comments from: M. van Oppen, J. Veron, H. Lessios, H. Guzman, M.
Takabayashi, T. Snell and O. Hoegh-Guldberg. I also wish to thank E. Bornham, C.
McNutt, J. Felsenstein, T. Hall, S. Kumar, D. Posada, M. Clement, and X. Xia.
Chapter II was made possible by a grant to G.M. Wellington from the
Environmental Institute of Houston, and from the support of G.E. Fox. Chapter III was
made possible by Sigma Xi grant in aid of research, and a grant to G.M. Wellington from
the National Geographic Society #6047-97. Chapter IV was made possible by grants to
Gerard M. Wellington from the Environmental Institute of Houston and the National
Geographic Society #6047-97.
iv
Phylogeny and Phylogeography of Porites & Siderastrea (Scleractinia: Cnidaria) Species in The Caribbean and Eastern Pacific; Based on The Nuclear Ribosomal ITS Region
--------------------------------------------- A Dissertation
Presented to
The Faculty of the Department of Biology and Biochemistry
University of Houston
---------------------------------------------
In Partial Fulfillment
Of the Requirements for the Degree
Doctor of Philosophy
---------------------------------------------
by
Zac Forsman
May 2003
v
DISSERTATION ABSTRACT
This study explores the ITS region (ITS-1-5.8S-ITS-2) as a genetic marker in two
prominent Scleractinian genera: Porites and Siderastrea, emphasizing the continuum
between population genetics and phylogenetics. Chapter I is a review and introduction.
Chapter II addresses widely-cited potential problems with the ITS region (intra-individual
heterogeneity and alignment gaps), and demonstrates how they can actually be
informative. Chapter III investigates a putative cryptic species; Porites lobata-Panama,
and examines the genetic structure and morphometric variability in P. lobata samples
collected from the Galápagos, Easter Island, Tahiti, Fiji, Rarotonga, and Australia.
Chapter IV examines shared ITS haplotypes in S. glynni and S. siderea, indicating that S.
glynni originated either from a recent passage through the Panamá canal, or through an
ancient (1-3mya) vicariant event. Both hypotheses have important implications for the
evolution of the ITS region. Chapter V is a summary of the major conclusions.
vi
CONTENTS List of Tables ............................................................................................................................................... vii
List of Figures ............................................................................................................................................. vii
I. Introduction and Background ............................................................................................................. 1 SIGNIFICANCE......................................................................................................................................... 1 PORITES.................................................................................................................................................... 3 RIBOSOMAL SPACERS........................................................................................................................... 5
II. Intra-species Variability And Alignment Gaps In The ITS Region Can Be Informative In Scleractinian Coral Families, Genera And Species; A Case Study In Porites, Siderastrea And Outgroup Taxa............................................................................................................................................ 11
DISCUSSION........................................................................................................................................... 30 III. Phylogeography and Morphological Variation in Porites lobata Across the Pacific: A Cryptic Panamanian species and Isolation Consistent with Ocean Currents. .................................................... 75
IV. The Siderastrea glynni (Scleractinia: Siderastreidae) Paradox: A Critically Endangered Species Or A Stowaway From The Caribbean? ITS Region Sequences Are Shared With S. siderea. 124
Reef-building corals form the structural foundation of one of the most diverse and
productive ecosystems on Earth. Corals have been a major component of reef
ecosystems since the late-Triassic (more than 200 million years ago). They have
persisted through several mass extinctions, as well as major global fluctuations in climate
and sea level. Despite the apparent long-term stability of reef corals, there is
widespread concern that human activity is linked to increasingly frequent episodes of reef
degradation. Hoegh-Guldberg (1999) reviewed widely cited anthropogenic threats to
coral reef ecosystems, including: land runoff, pollution, terrestrial pathogens, over fishing
of herbivores, & coral bleaching (minor increases in ocean temperature dissociates the
coral/algal symbiosis resulting in mortality). Global warming trends are correlated with
mass coral bleaching events and increased levels of atmospheric CO2 may adversely
affect calcification and growth rates.
The sensitivity of corals to minor environmental changes is a valuable source of
information. Corals are indicator species, yielding information about the present status
of ecosystem health. Corals also yield a great deal of information about the past.
Coral are long-lived sedentary clonal organisms that secrete calcium carbonate skeletons
in annual growth bands analogous to tree-rings. Coral proxi-records are central to our
understanding of past climates, changes in sea level, and patterns of biogeography,
(reviewed in Romano et al. 2000). Corals are ancient organisms, descendants of one of
2
the first mulitcellular animals on Earth. Cnidarians (the phylum to which corals are a
member) represent one of the most important transitions in metazoan evolution. They
are the first animals with layers of specialized tissues, which allowed the first appearance
of evolutionary inventions such as: the gastric cavity, movement, muscles, nervous tissue,
and photoreception. Just as the physical skeleton has been a source of proxi-records in
recent geologic history, the genome is a valuable proxi-record of evolutionary
relationships.
Genetic studies have great potential to clarify one of the largest problems in coral
reef studies; many coral species are difficult to identify at the species level. The species
is one of the most fundamental and important units in the study of biology. Without the
ability to distinguish between species, it is impossible to recognize species ranges and
boundaries, dispersal among populations, or interactions between species. With no
ability to recognize species, one cannot determine which populations are endangered, or
even recognize when extinction occurs.
Coral species are difficult to define for several reasons: (1). Convergent
evolution: morphological taxonomic characters are often as variable within a species as
between species. Morphologically indistinguishable species could be closely related
"sibling species", or more distantly related "cryptic species" (after Knowlton 1993, 2000).
(2). Phenotypic plasticity: some species are broadly adapted to a wide range of habitats,
and exhibit different ecomorphs in response to different environmental conditions
(examples in Veron 1995, 2000). (3). Hybridization and reticulate evolution: Mass
spawning produces opportunities for hybridization between species because many corals
3
spawn simultaneously. Some corals are long-lived at the colony level (hundreds of
years or more), and geographically widespread. Changes in ocean circulation may
introduce genetically and morphologically disparate populations, or create opportunities
for hybridization between 'species' vis-à-vis Veron's (1995) theory of reticulate evolution
by sea surface vicariance.
PORITES
The genus Porites Link 1807 has been one of the most important, widespread and
abundant reef-building corals over the last 20 million years (Frost 1977). Porites occurs
worldwide in the tropics, it has the largest range (Veron 1995, 2000) and has one of the
highest estimated dispersal abilities of any extant coral genera (Fadlallah 1983).
Despite the importance of Porites in coral reef ecosystems, relationships between species,
or populations within species remain largely unknown. Progress in Porites systematics
has been slow because it is difficult to determine what constitutes a 'species' within this
genus.
Taxonomy in Porites is based on morphological and skeletal architecture and is
renowned as among the most difficult and in the most need of revision. In Porites,
corallites are very small, irregular, perforated and may be as highly variable within a
single colony as between species. Colony form is also highly variable, for example P.
lobata ranges from encrusting, plate-like or bolder-like forms, to thin protruding lobe, fin
or columner forms. Many morphological differences can be attributed to a
phenotypically plastic response to environmental conditions (available light, water
motion, predation, etc.), while others may be indicative of underlying genetic variation.
4
These highly variable and hard to measure characteristics make it very difficult to divide
Porites into discrete species.
Around 122 Porites species have been named, although many of these names are
considered invalid (Veron 1986). Cairns (1999) recognizes 41 species as valid. Six
species are recognized in the Caribbean; P. porites, P. furcata, P. divaricata, P.
astreoides, P. colonensis and P. branneri. In the far eastern Pacific, 8 species are
currently considered valid; P. lobata, P. panamensis, P. rus, P. arnudi, P. australiensis,
P. lutea, P. lichen and P. sverdrupi. (The latter appears so similar to P. panamensis, that
it may not be a separate species (Veron personal communication)).
Early studies of Porites relied entirely on morphology, (Bernard 1902; Brakel
1977). Bernard (1906) abandoned the Linnean classification system entirely for this
genus, and used a system of numbers. Brakel (1977) concluded that patterns of
morphological Porites are so complex that no simple taxonomic resolution is possible at
the species level. His analysis suggested that P. astreoides and P. porites represented
only the most opposite extremes of recognizable ‘phenons’ in a continual gradient that he
attributed to diversifying selection. The authors maintained that Porites typified a
'species problem' in coral, due to countless intermediate forms.
Garthwaite et al. (1994) used multiple allozyme loci to determine that some
Porites species were genetically distinct. Wiel (1992a, 1992b) through allozymes and
multivariate morphometric statistics distinguished 8 putative species on both sides of the
Isthmus of Panamá. Weil concluded that a large portion of the morphometric variation
5
can be attributed to genetic variation, and that the recognizable species around the
Isthmus of Panamá are likely to be discrete entities.
Hunter (1988) also used allozymes to examine genetic structure in the Hawaiian
endemic P. compressa. Hunter et al. (1997) were among the first to use DNA in a
species level study of Scleractinia. Most molecular techniques are difficult to apply to
corals, because they can be contaminated by symbiotic algae (zooxanthellae). Hunter et
al. (1997) used the Internal Transcribed Spacers of ribosomal RNA genes to examine
Hawaiian and Floridian Porites species and their algal symbionts. The marker
distinguished between species, and revealed species level polymorphisms within
Hawaiian P. lobata.
RIBOSOMAL SPACERS
Nuclear ribosomal genes are a mosaic of variability, and are therefore useful for a
broad range of comparative studies. Regions that have species polymorphisms flank
others that are highly conserved across phyla. This allows studies at different levels of
taxonomic resolution. Primers can be designed from phylogenetically conserved
regions that bridge the gap across highly polymorphic regions. Eukaryotic rDNA
consists of tandemly repeated clusters of the 18S, 5.8S, and 28S genes separated by two
internally transcribed spacers, ITS-1 and ITS-2 (see Figure I-1). Levels of
polymorphism roughly correspond with taxonomic levels ranging from phyla (18S) to
species and below (ITS) (Hillis and Dixon 1991). Studies in a wide variety of
organisms have demonstrated that the spacers are useful for species level phylogeny,
6
species identification, and examining hybridization between species (reviewed in Chapter
II). Ribosomal genes are the largest and most ancient multigene family, occurring in
tandem repeats hundreds or thousands of copies long. The genes and the spacers
between them are usually orders of magnitude less variable within a species than between
species.
The ITS region shows variability within species as well as differences between
them, therefore it has great potential as a genetic marker in a wide variety of studies.
The marker also has serious potential drawbacks, such as polymorphism (sometimes at
high levels) within a single genome. Empirical studies of ribosomal spacers are needed
to further understand the nature of the forces that homogenize them within an
interbreeding lineage, and to determine where population processes such as interbreeding
end and divergence and speciation begin.
7
FIGURES
Figure I-1
A diagram of a Eukaryotic ribosomal operon, which consists of tandem repeats of
ribosomal genes and spacers: IGS, is the intergenic spacer located between each set of
ribosomal genes. The 18S is followed by ITS-1, the 5.8S and the ITS-2, followed by the
28S. Also shown are the relative locations of the 'universal Eukaryotic' PCR primers
used in this study ITS-1, and ITS-4 (White et al. 1990).
8
Figure I-1
9
LITERATURE CITED Bernard, H. M. (1902). The species problem in corals. Nature 65, 560. Brakel, W. H. (1977). Corallite variation in Porites and the species problem in corals. Proc. Third Intl. Coral Reef Symp. Miami, p 457-462. Cairns, S. D., Hoeksema, B. W. and Van Der Land, J. (1999). Appendix: List of Extant Stony Corals. In Atoll Research Bulletin, vol. 459. Washington, D.C.: Smithsonian Institution. Fadlallah, Y. H. (1983). Sexual reproduction, development and larval biology in Scleractinian corals: a review. Coral Reefs 2, 129-150. Frost, S. H. (1977). Miocene and Holocene evolution of Caribbean province reef-building corals. Proc. Third Int. Coral Reef Symp., Maiami 2, 353-359. Garthwaite, R. L., Potts, D. C. and Done, T. J. (1994). Electrophoretic identification of Poritid species (Anthozoa: Scleractinia). Coral Reefs , 49-56. Hillis, D. M. and Dixon, M. T. (1991). Ribosomal DNA: Molecular Evolution and Phylogenetic Inference. Quart. Rev. Bio. 66, 411-453. Hoegh-Guldberg, O. (1999). Climate change coral bleaching and the future of the world's coral reefs. Marine and Freshwater Research 50, 839-66. Hunter, C. L. (1988). Genotypic diversity and population structure of the Hawaiian reef coral Porites compressa, Ph.D. Dissertation. University of Hawaii . Hunter, C. L., Morden, C. W. and Smith, C. M. (1997). The utility of ITS sequences in assessing relationships among zooxanthellae and corals. Proc. 8th int coral reef sym. , 1599-1602. Knowlton, N. (1993). Sibling species in the sea. Annu. Rev. Ecol. Syst 24, 189-216. Knowlton, N. (2000). Molecular genetic analysis of species boundaries in the sea. Hydrobiologia 420, 73-90. Link, H. F. (1807). Bescheibung der Naturalein. Sammlungen der Universaitat Rostock, 3, 161-165.
10
Romano, S. L. and Cairns, S. D. (2000). Molecular phylogenetic hypotheses for the evolution of Scleractinian corals. Bull. Mar. Sci. 63, 1043-1068. Veron, J. (1995). Corals in space and time; the biogrography and evolution of the Scleractinia. London: Cornell. Veron, J. E. N. (1986). Corals of Australia and the Indo-Pacific, pp. 644. New York: Angus and Robertsons Publishers. Veron, J. E. N. (2000). Corals of the World, vol. 3 (ed. M. Stafford-Smith). Townsville, Australia: Australian Institute of Marine Science. Weil, E. (1992). Genetic and morphological variation in Caribbean and eastern Pacific Porites (Anthozoa, Scleractinia), preliminary results. Proc 7th Int. Coral Reef Sym. Guam 643-656. Weil, E. F. (1992). Genetic and morphological variation in Porites (Cnidaria, Anthosoa) across the Isthmus of Panama. Ph.D. Dissertation. pp. 327. Austin TX: University of Texas. White, T. J., Gruns, T. L. and Taylor, W. J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A guide to methods and applications (ed. Innis, D.H.;Sninsky,J.J.;White,T.J.). San Diego: Academic Press.
11
II. Intra-species Variability And Alignment Gaps In The ITS Region Can Be Informative In Scleractinian Coral Families, Genera And Species; A Case Study In Porites, Siderastrea And Outgroup Taxa.
ABSTRACT In this study, we use an empirical example to examine two of the most widely
acknowledged problems with the ITS region as a phylogenetic marker: intra-species variability, and alignment ambiguities resulting from insertions and deletions. Several sequences from each individual were examined from the following Porites and Siderastrea species; P. lobata, P. lobata-panama (a genetically distinct lineage that may represent a new cryptic species), P. astreoides, P. colonensis, P. sverdrupi, P. panamensis, P. divaricata, P. rus, P. furcata, S. stellata, S. radians, and S. siderea. A phylogeny was then estimated including four outgroup sequences from the GenBank database (Tubastrea, Balanophyllia, Scapophyllia and Montastrea). Intragenomic variation in all species sampled was low. In Porites and Siderastrea sequences, nucleotide diversity was significantly lower within a population, than between populations separated by thousands of kilometers (averaging 0.9%±0.5 and 1.2%±0.5 respectively), indicating that geographic structure may exist. The average difference between species was at least one order of magnitude higher (12.0%±1.2). These results indicate that the ITS region is an informative marker at the species-level and below.
Despite a patchwork of conserved sequence motifs among Scleractinian families, genera and species, numerous insertions and deletions make objective sequence alignment problematic. The effects of alignment gaps on phylogenetic estimates were examined by systematically permuting gap penalties to generate 50 alternative alignments. A maximum likelihood tree was then constructed for each alternative alignment. The trees were remarkably congruent, with the majority of nodes supported by all of the alternative alignments. The same general topology (although much less resolved) was also supported by removing all of the alignment gaps. Alignments at opposite ends of the gap penalty spectrum had unusual ts/tv (transition/transversion) ratios, high discrepancies between substitution and gap distance, and unique nodes. Alignments with mid-range gap penalties had ts/tv ratios similar to conserved portions of the alignment, high character congruence between substitutions and gaps, and the most congruent tree topology. The mid-point alignment was chosen to estimate a phylogeny with maximum likelihood, maximum parsimony, and neighbor-joining methods. The data did not significantly deviate from expectations of a molecular clock at the genus level and below. The phylogeny is consistent with several previous molecular and paleontological studies. This study represents the first molecular phylogeny at the family to species level in Scleractinia.
12
INTRODUCTION
In reef building Scleractinian corals, high levels of genetic and or morphological
variation have resulted in a great deal of taxonomic confusion and controversy.
Intermediate and overlapping morphologies are thought to be due to convergent or
parallel evolution, or by introgression from distinct lineages in 'hybrid species
complexes', resulting in non-discrete patterns of genetic and or morphological variation
(Veron 1995; Lopez and Knowlton 1997; Knowlton 2000; van Oppen et al. 2000, 2002).
This problem is not limited to Scleractinia, but pertains to many of the earliest branches
of the tree of life, where discrete genetic and morphological boundaries are often unclear.
A second major problem in coral systematics is that many of the well-studied and
widely used molecular markers have low levels of polymorphism. Mitochondrial DNA
evolves at a slow rate in corals relative to other Metazoans, such as vertebrates (Romano
and Palumbi 1996,1997; Shearer et al. 2002; van Oppen 1999). DNA repair
mechanisms that are present in free-living relatives of mitochondria appear to be retained
in coral mitochondria, which is a likely explanation for the low levels of polymorphism
(van Oppen 1999). Molecular markers such as the 28S nuclear ribosomal gene, and the
mitochondrial 16S ribosomal gene, have been used for establishing relationships between
orders and families; (Romano and Palumbi 1996,1997; Veron et al. 1996; Chen et al.
1995) however, are not informative at the genus level and below.
The transcribed spacers of nuclear ribosomal genes (ITS-1 and ITS-2), are
becoming one of the most widely used molecular markers at the species level and below
13
in Scleractinian coral (Hunter et al. 1997; Lopez and Knowlton 1997; Odorico and Miller
1997; Medina et al. 1999; van Oppen et al. 2000, 2002; Diekmann at al. 2001;
Takabayashi et al. 1998a, 1998b; Rodriguez-Lanetty and Hoegh-Guldberg 2002;
Márquez et al. in press). There is a considerable precedent for the use of ITS to infer
relationships at or below the species level in a wide variety of other taxonomic groups.
It is widely used for identifying cryptic species of medically or commercially important
fungi (for example; McCullough et. al. 1998; Kuninaga et al. 1997; Arlorio et al. 1999).
It is frequently used in plant systematics (reviewed in Baldwin et al. 1995) and to reveal
relationships in species complexes (Jeandroz et al. 1997; Hsiao et al. 1995; Sang et
al.1995; Wen and Zimmer 1996). It has also been used to reveal geographic
polymorphisms and species relationships in insects (Wesson et al. 1993; Marcilla et al.
2001) and a variety of marine organisms; e.g. deep sea hydrothermal vent polycheates
(Jollivet et al 1995), the globally distributed green algae Chlorophyta (Bakker et al.
1995), the ahermatypic coral Balanophyllia elegans (Beauchamp and Powers 1996) and a
corallimorpharian anemone Rhodactis (Chen and Miller 1996).
Despite the wide use of the ITS region in phylogenetic studies, many authors have
acknowledged two major problems with the marker that can severely confound
phylogenetic studies: (1). Intragenomic variability can be quite large in some species,
pseudogenes or separate chromosomal lineages can make phylogenetic estimation
problematic. (2). hyper-variable portions of ITS-1 and ITS-2 are prone to numerous
insertions and deletions, which can result in alignment ambiguities. Distantly related
species, or species from different genera or families become nearly impossible to align
14
objectively, despite the existence of patches of conserved sequence motifs. These two
problems are addressed in more detail in the following separate sections.
Intragenomic variability
Within a typical Eukaryotic genome there are hundreds, or thousands of copies of
ribosomal genes, which are separated by rapidly evolving spacer sequences. It has been
observed that the spacer sequences tend to be considerably more similar within
reproductive groups, than between separate species (Learn and Schaal 1987, Coleman
and Mai 1997). Concerted evolution, is a process that homogenizes tandem gene
repeats such as ribosomal genes and spacer sequences. Unequal crossover and gene
conversion during crossover are the two most widely accepted mechanisms for concerted
evolution (Dover 1982). Unequal crossover is due to tandem gene repeats occasionally
mis-pairing, resulting in one gamete with extra copies and one with fewer. Gene
conversion is a process whereby one allele is converted to another by cellular repair
mechanisms, which also occurs during recombination. Recombination does not occur
between reproductively isolated individuals; therefore, non-conserved sequences are free
to rapidly diverge after speciation (Elder and Turner 1995). Ribosomal spacer gene
trees usually closely reflect the species tree, provided that the rate of turnover (gene
conversion and unequal crossover) is greater then the rate of speciation (Hillis and Dixon
1991). In other words, the variability within an individual or species must be low
relative to the average difference between closely related species in the taxonomic group
of interest.
15
Intragenomic variability is usually attributed to one of several causes. The
existence of extremely divergent paralogues genes within a genome is usually associated
with the presence of inactive pseudogenes. Divergent pseudogenes have been
associated with ancient hybridization events between separate species, which can result in
polyploidy, followed by chromosomal inactivation (Wendel et al. 1995; Sang et al. 1995;
O'Donnell and Cigelnik 1997; van Oppen et al. 2000; Muir et al. 2000). Some
taxonomic groups have several active arrays of ribosomal genes (nucleolus organizer
regions) located on separate chromosomes. Moderately divergent intra-individual
paralogues have been associated with slower rates of crossover and gene conversion
between these separate chromosomal lineages (Arnheim et al. 1980; Polanco et al. 2000).
Relatively low levels of intra-specific nucleotide diversity make phylogenetic and
population studies much less problematic; however, even if nucleotide diversity is low,
separate species cannot easily be distinguished if speciation occurs faster than the rate of
concerted evolution.
Population level processes are also likely to have an important role in influencing
the homogeneity of ribosomal spacers within a species. A highly subdivided species
with isolated populations might be expected to have higher ribosomal spacer
heterogeneity then a species with no subdivision. Isolated populations are likely to
undergo genetic drift, because concerted evolution is maintained by recombination, and
net recombination will be less frequent between isolated populations. In order for the
ITS region to be useful for population genetic studies, a hierarchy of variability must
exist whereby average nucleotide diversity is significantly lower at the intragenomic level
16
then the intrapopulation level, which is in turn lower than the interpopulation level. If
such patterns exist, then the ITS region could be useful as an indicator of relative gene
flow between populations. Empirical studies that examine the variability of ribosomal
spacers from the population to species level processes are necessary to gain an
understanding of ITS region population and evolutionary dynamics.
Alignment ambiguity
A multiple sequence alignment is a single hypothesis about how a given set of
sequences has evolved. Alignment can have a greater effect on phylogenetic estimation
than the tree making method (Morrison and Ellis 1997). Multiple sequence alignment is
generally not problematic for closely related sequences or highly conserved sequences,
where the majority of mutations are substitutions. In relatively non-conserved
sequences such as introns or ribosomal spacers, a large percentage of the mutations are
likely to be insertions and deletions. Since it is usually not possible to determine
whether an alignment gap between two sequences was the result of an insertion or a
deletion, these events are referred to as indels. Indels generally originate during
replication, recombination, or transposition. The occurrence of gaps in a given set of
sequences usually follows a bimodal distribution consisting of large and small gaps.
Small gaps usually consist of simple repeats resulting from replication slippage, whereas
large gaps tend to result from recombination or transposition (Li 1997). The more gaps
there are in an alignment, the higher the number of possible alternative alignments, and
the higher the number of ambiguous positions. Ambiguous positions provide the
17
opportunity for the subjective judgment of a researcher to consciously or unconsciously
bias the result. The more distantly related the sequences the greater the chance of indel
saturation, resulting in greater homoplasy (contradictory data due to reversals, convergent
evolution, or parallel evolution).
There are several widely employed methods of handling gaps in sequence
alignments. The most commonly applied approach is to "manually improve" an
alignment after its initial construction by a computer algorithm. The goal of manual
"improvement" is to increase the apparent similarity between sequences in the alignment,
however, clear objective criteria for basing such "improvements" are often lacking
(Giribet and Wheeler 1999). This can be especially problematic in hyper-variable
sequences such as introns or transcribed spacers. Although a manually improved
alignment may appear "better" then a computer generated alignment, the appearance
could be misleading, and poorly reflect how the molecule actually evolved. A manual
alignment is unlikely to take into account that some substitutions have higher
probabilities of occurring then others in a given data-set (e.g. the bias for transitions over
transversions).
A second strategy that is often employed is to remove strips of the sequence
alignment that contain alignment gaps altogether (Olsen and Woese 1993). The obvious
setback of this approach is that a great deal of valuable, and potentially informative data
becomes lost. It has been demonstrated that gaps can contain phylogenetic signal
(Giribet and Wheeler 1999), and ignoring characters from a phylogenetic analysis can be
subjective. A third, less widely used, approach is to generate and compare several
18
alternative alignments (Morrison and Ellis 1997; McFadden et al. 2001), or to search for
optimal alignments by parsimony criteria (Wheeler and Gladstein 1988). These
promising approaches are more computationally expensive, and cannot guarantee that the
entire alignment space has been examined, or that the 'correct' alignment can even be
found. However, each approach has the benefit that they are not subjective, and can
give an indication how much homoplasy is present in the data set. Presumably, a strong
underlying phylogenetic signal will reflect the same relationships under a wide variety of
alignment conditions.
The goals of this study are: (1) to examine the hierarchical nature of variability in
the ribosomal spacer region from the individual to species levels. (2) to examine the
phylogenetic signal from species to family level through comparisons of many alternative
alignments generated by the permutation of gap penalties, and (3) to examine the
relationships between several prominent species, genera and families of Scleractinian
coral.
METHODS
Small, fragments, ca. 10-15 grams of tissue and skeleton were removed from
colony edges, branches, or protuberances. Samples were collected at least 10 meters
apart to avoid collecting colonies that originated from fragmentation or budding.
Samples were preserved in 95-100% ethanol. The samples were divided into several
pieces in the laboratory, a small piece was stored in fresh ethanol at -20°C for genetic
analysis, and larger pieces were placed in bleach to dissolve the soft tissue, prior to
19
drying. Voucher specimens, and scaled digital microscope images were collected for the
majority of specimens and will be made available for other studies upon request. Table
II-1 summarizes the geographic location of the samples collected, the collector and the
date of collection.
DNA extraction, PCR, Cloning and Sequencing
Many authors have reported that extracting DNA from Scleractinia can be
problematic. Mucous, polysaccharides, pigments, or other DNA co-precipitates are
often cited as inhibiting the PCR reaction. After a trial of many widely available
extraction protocols, the following protocol consistently yielded the best results. A few
milligrams of tissue and skeleton were dried in a vacuum centrifuge for 20min, the
sample was then homogenized in a solution of 250µl of 50mM tris-HCL (pH 8.0) and
10mM EDTA with a micro-pestle for 2 to 5 minutes. The homogenate was then
frequently inverted during a 5 minute room temperature incubation in 250µl of 20mM
NaOH and 1% SDS. A volume of 350µl of 3.0M potassium acetate (pH 5.5) was added
to the mixture and incubated for 5 minutes on ice followed by centrifugation at maximum
speed. The top 500µl of the cleared lysate was then transferred to a new tube and the
DNA was precipitated by centrifugation in 1ml isopropanol. The sample was then
washed with 70% EtOH, dried and resuspended in 200µl of H2O.
The nuclear ribosomal internal transcribed spacer region (spanning a partial
sequence of the 5’ end of the 18S gene, the complete sequence of ITS-1, 5.8S gene and
ITS-2, and a partial sequence of the 3’ end of the 28S gene) was amplified using the
Matrix of averaged genetic distance between species (in substitutions per site,
calculated by the Kimura 1980 method), and standard errors. The first column
represents average intra-species differences. Distances were calculated including the
entire ITS region (ITS-1, 5.8S, and ITS-2). Standard errors were estimated by 500
bootstrap replicates implemented in MEGA 2.1 (Kumar et al. 2001). Inter-species
distance was calculated from an alignment (GOP=0.2, GEP=0.1) that included all 130
sequences.
41
Table II-2
species Intra-species S. siderea S. radians S. stellata P. astreoides P. divaricata P. furcata P. sverdrupi P. panamensisP. rus P. colonensis P. lobata- Panama
D.) Siderastrea species Ts/Tv 1.72 E.) 5.8S only Ts/Tv= 1.32
56
Figure II-8
The relationship between gap distance and substitution distance between the
permuted alignments. GOP and GEP refer to Gap Opening Penalty and Gap Extension
Penalty. The slope of the line indicates that the alignment with largest gap penalties
(GOP=8.0, GEP=4.0) had approximately 2 substitutions for every alignment gap, the
mid-point alignment (GOP=2.0, GEP=1.0) had 1 substitution for every 1 gap, and the
alignment with lowest gap penalties (GOP=0.01, GEP=0.01) resulted in 0.71
substitutions for every gap. The r2 and p value indicate that the regressions are highly
significant.
57
y = 0.71x + 0.01R2 = 0.84p<0.001
y = 1.14x + 0.02R2 = 0.75p<0.001
y = 1.9x + 0.04R2 = 0.67p<0.001
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4Gap Distance
Subs
titut
ion
Dis
tanc
e (p
)
O 8.0,E 4.0
O 2.0,E 1.0
O .01,E .01
GOP=8.0GEP=4.0
GOP=2.0GEP=1.0
GOP=0.01GEP=0.01 r2
r2
r2
Figure II-8
58
Figure II-9 The 'Mid-point' alignment (GOP=2.0, GEP=1.0), maximum parsimony and
maximum likelihood consensus tree. Abbreviations are as in Figure II-4. Values at the
nodes are bootstrap values (500 replicates); regular script indicates maximum parsimony,
italic script indicates maximum likelihood, and bold script indicates support by both
methods. The alignment had 278 sites that were parsimony informative sites (gaps were
counted as a fifth character state, although if gaps were ignored the same topology
resulted), a heuristic branch and bound search yielded two parsimonious trees, CI=0.77,
RI=0.83. The topology was identical in both methods, with the exception of the node
indicated with an asterisk*. Balanophyllia-Tubastraea were grouped with Siderastrea
in the maximum likelihood topology.
59
Figure II-9
P. rus
P. lobata-panama
P. lobata G
P. lobata A
P. astreoides B
P. astreoides Z
P. colonensis
P. divaricata
P. furcata
P. sverdrupi
P. panamensis
Balanophyllia
Tubastrea
S. stellata
S. radians
S. siderea A
S. siderea B
Montastrea
Scapophyllia
100
10099
10067
5074
100
100 100
100
100
100 73*
100
100
10098
100
10094
60
Figure II-10 A Neighbor-Joining phylogram of all of the sequences collected for this
study (130) and from the database (4), of an alignment constructed using the 'mid-point'
alignment gap penalties. Positions with insertions/deletions were included in the
analysis, the 5.8S gene was invariant among Porites taxa and was therefore excluded,
leaving 981 remaining positions. Distances were calculated with the Kimura (1980)
method, with 1000 bootstrapped replicates in Mega 2.1 (Kumar et al. 2001), bootstrap
values less then 70% are not shown. The width of each triangle base is proportional to
the number of sequences in the clade (approximately 4 pixels/taxon). The height (depth
in time) of the triangle is proportional the variability within the group. The scale is
proportional to number of nucleotide substitutions per site, and estimated time of
divergence assuming a constant rate of 0.004 substitutions per site per million years
(Savard et al. 19993). The large shaded rectangle indicates an area where distance is
likely to be underestimated, Felsenstein's (1988) molecular clock hypothesis can be
rejected for the 'outgroup' taxa. The relationship between substitutions and time do not
significantly deviate from expectations of linearity For the 'ingroup' taxa. A light gray
rectangle represents a period of mass extinction (1-4 mya), dark gray lines indicate
important dates: 3.5-3.8 mya = complete closure of the Isthmus of Panamá (Kegwin
1982) , 1.6-2.5 mya = the first appearance of P. divaricata in the fossil record (Budd et
al. 1994).
61
Figure II-10
Late Cretaceous
Palaeocene
Eocene
Oligocene
Miocene
Pleistocene
P. lobata
P. lobata-panama P. rus
P. astreoides
P. panamensis-sverdrupi
P. furcata P. divaricata
P. colonensis Balanophyllia Tubastrea S. radians S. stellata
S.siderea
Montastrea Scapophyllia
99
99
86
7699
85
99
99
83
75
9999
9999
99
91
9994
83
99
0.00.10.2 0.3 percent divergence
05101520253035404550 55 60 65 70 75 MY
Pliocene
62
Figure II-11
The 'Mid-point' alignment GOP=2.0, GEP=1.0, of the representative sequences
used for examing alignment parameters. Gaps are shaded gray, the relative positions of
the 18S, ITS-1, 5.8S, ITS-2, and 28S are indicated below the alignment.
63
18S 3' end ITS-1 5' begin
Figure II-11
64
Figure II-11 continued
ITS-1 3' end 5.8S 5' begin
5.8S 3' end ITS-2 5' begin
65
Figure II-11 continued
ITS-2 3' end 28S 5' begin
66
Figure II-12
An illustration of several taxonomists (past and present) synthesis of the
Scleractinian fossil record. Abbreviations are as follows: P= Poritidae, D=
Dendrophylliidae, S=Siderastreidae, F=Faviidae, and M=Merulinidae. Bold lines
indicate the depth of the family fossil record; dashed lines represent inferred relationships
with extinct or living families. Brackets indicate that the author placed the taxa in
close proximity. Shaded gray rectangles mark periods of mass extinction and species
turnover in the fossil record. The geologic periods are not drawn to scale. The ITS
data significantly deviates from expectations of a molecular clock at the genus and family
level, and is likely to underestimate divergence times. The data is displayed for the sake
of comparison.
67
Palaeocene
Eocene
Oligocene
Miocene
Late Jurassic
Mid Jurassic
Early Jurassic
Late Triassic
Early Cretaceous
mid Cretaceous
late Cretaceous
(P D ) (M, F) (S)
Veron 2000, 1996
(P ( D, S ) (M, F) (P , S ) (M, F) (D) (P , S ) (M, F) (D)
Wells 1956 Roniewicz and Morycowa (1989)
ITS Region
Figure II -12
68
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Muir, G. C., Fleming, C. and Schlotterer, C. (2001). Three divergent rDNA clusters predate the species divergence in Quercus petrea and Quercus robur. Mol. Biol. Evol. 18, 112-119. O'Donnell, K. and Cigelnik, E. (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7, 103-16. Odorico, D. M. and Miller, D. J. (1997). Variation in the Ribosomal Internal Transcribed Spacers and 5.8S rDNA Among Five Species of Acropora (Cnidaria;Scleractinia): Patterns of Variation Consistent with Reticulate Evolution. Mol. Biol. Evol. 14, 465-473. Olsen, G. J. and Woese, C. R. (1993). Ribosomal RNA: a key to phylogeny. (review). The FASEB Journal 7, 113-23 bibl il. Polanco, C., Gonzalez, A. I. and Dover, G. A. (2000). Patterns of variation in the intergenic spacers of ribosomal DNA in Drosophila melanogaster support a model for genetic exchanges during X-Y pairing. Genetics 155, 1221-9. Rodriguez-Lanetty, M. and Hoegh-Guldberg, O. (2002). The phylogeography and connectivity of the latitudinally widespread scleractinian coral Plesiastrea versipora in the Western Pacific. Mol. Ecol 11, 1177-1189. Romano, S. L. and Cairns, S. D. (2000). Molecular phylogenetic hypotheses for the evolution of Scleractinian corals. Bull. Mar. Sci. . Romano, S. L. and Palumbi, S. R. (1996). Evolution of Scleractinian corals inferred from molecular systematics. Science 271, 640-642. Romano, S. L. and Palumbi, S. R. (1997). Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in Scleractinian corals. J Mol Evol 45, 397-411. Roniewicz, E. and Morycowa, E. (1993). Evolution of the Scleractinia in the light of microstructural data. Cour Forsh Inst Senckenberg 164, 233-240. Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425. Sanderson, M. J. and Shaffer, H. B. (2002). Troubleshooting molecular phylogenetic analyses. Annu. Rev. Ecol. Syst. 33, 49-72.
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Sang, T., Crawford, D. J. and Stussy, T. F. (1995). Documentation of reticulate evolution in peonies (Paeonea) using internal transcribed spacer sequences of nuclear ribosomal DNA: implications for biogeography and concerted evolution. Proc. Natl. Acad. Sci. USA 92, 6813-6817. Savard, L., Michaud, M. and Bousquet, J. (1993). Genetic diversity and phylogenetic relationships between birches and alders using ITS, 18S rRNA and rbcL gene sequences. Mol Phylogenet Evol 2, 112-8. Schlotterer, C., Hauser, M. T., von Haeseler, A. and Tautz, D. (1994). Comparative evolutionary analysis of rDNA ITS regions in Drosophila. Mol Biol Evol 11, 513-22. Shearer, T. L., Van Oppen, M. J., Romano, S. L. and Worheide, G. (2002). Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria). Mol Ecol 12, 2475-87. Swofford, D. L., Olsen, G. L., Waddell, P. J. and Hillis, D. M. (1996). Phylogenetic inference. In Molecular systematics (ed. D. M. M. C. Hillis, Mable B.K). Sunderland MA: Sinauer Associates. Takabayashi, M., Carter D. A. Loh W. K. Hoegh-Guldberg. (1998). A coral-specific primer for PCR amplification of the internal transcribed spacer region in ribosomal DNA. Mol Ecol 7, 928-30. Takabayashi, M. and et al. (1998). Inter-and Intra-Specific Variability in Ribosomal DNA Sequence in the Internal Transcribed Spacer Region of Corals. Proc. Of the Aust. Coral Reef Soc. 75 Ann. Conf. , 241-248. Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680. van Oppen, M. J., Willis, B. L. and Miller, D. J. (1999). Atypically low rate of cytochrome b evolution in the scleractinian coral genus Acropora. Proc R Soc Lond B Biol Sci 266, 179-83. van Oppen, M. J. H., Willis, B., van Rheede, T. and Miller, D. J. (2002). Spawning times, reproductive compatibilities and genetic structuring in the Acropora aspera group: evidence for natural hybridization and semi-permeable species boundaries in corals. Mol. Ecol. 11, 1363-1376.
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75
III. Phylogeography and Morphological Variation in Porites lobata Across the Pacific: A Cryptic Panamanian species and Isolation Consistent with Ocean Currents.
ABSTRACT Gradual morphologic differences occur between geographic regions in the coral
species P. lobata. This has led to considerable taxonomic controversy; some authors contend that each distinct morphology represents a genetically distinct entity; others suggest that each form is the result of a phenotypic response to environmental conditions. An alternative hypothesis is that genetic and phenotypic cohesiveness are directly related, and maintained by ocean surface currents. Here we examine genetic variability of the ITS region in P. lobata from Panamá, Galápagos, Easter island, Tahiti, Fiji, Rarotonga and Australia. We examine morphometric variability in a subset of these populations (Panamá, Galápagos, Easter Island, Tahiti, and Fiji).
We report a putative cryptic species of P. lobata in Panamá. The species is reciprocally monophyletic according to the ITS region, and morphologically distinct according to a principal component discriminant analysis of corallite level characteristics, which allows 95% of all corallites to be classified as distinct from P. lobata. We designate this putative new species P. lobata-panama. Across the rest of the range of P. lobata, the discriminant analysis indicates that that a large portion of the variance is due to differences between regions, with small differences occurring between neighboring regions, and the largest differences occurring between geographic extremes. An AMOVA (Molecular Analysis of Variance) indicates that a significant portion of the genetic variance is due to differences between populations. Easter Island is the most isolated population, and is the most genetically and morphometrically distinct. A Nested Clade Analysis of the ITS region haplotypes rejects the null hypothesis of no association between phylogenetic and geographic structure. Isolation by distance exists for at least two separate clades that contain the Easter Island haplotypes. The relationships between many of the morphologic traits are significantly correlated to genetic relationships according to Mantel tests of the distance matricies. Gradual genetic and morphometric variation between the geographic regions are consistent with the expectations of recurrent gene flow and isolation by distance. The genetic and morphometric relationships between geographic regions are consistent with observations of Pacific Ocean surface currents derived from satellite data.
76
INTRODUCTION
The genus Porites (Link 1807) has been one of the most important, widespread
and abundant reef-building corals over the last 20 million years (Frost 1977). Porites
occurs worldwide in the tropics. Porites species have some of the highest dispersal
potentials (Faldallah 1983) and largest geographic ranges (Veron 1995, 2000). Despite
the importance of Porites in coral reef ecosystems, relationships between species, or
between populations remain largely unknown. Progress in Porites systematics has been
slow because it is difficult to determine what constitutes a 'species' within this genus.
Taxonomy in Porites is based on morphological and skeletal architecture and is
renowned as among the most difficult and in the most need of revision. In Porites,
corallites are very small, irregular, perforated and highly variable. Variability in colony
and corallite level skeletal characteristics is typified in the most cosmopolitan Porites
species P. lobata (Dana 1846). P. lobata occurs in a wide variety of habitats over an
enormous geographic range, spanning across the Pacific and Indian oceans. Colony and
corallite level characteristics have been observed to vary geographically, which has led to
numerous synonyms, and named 'formae' and 'subformae' (Bernard, 1902; Vaughan,
1907; Veron and Pichon, 1982; Veron 1995, 2000). Colony form ranges from
encrusting, plate-like or bolder-like forms, to thin protruding lobe, fin or columner
morphology. Some authors have maintained that these morphological differences are
the result of a phenotypically plastic response to environmental conditions (available
light, water motion, predation, etc.), while others suggest that the variation reflects
isolation between genetically distinct populations, or even separate species.
77
It is generally difficult to define coral species for several reasons: (i).
Convergent evolution: morphological characters in Porites are often nearly as variable
within an individual as between species. Morphologically indistinguishable species
could be closely related "sibling species", or more distantly related "cryptic species"
(after Knowlton 1993). (ii). Phenotypic plasticity: some species are broadly adapted to
a wide range of habitats, and exhibit different ecomorphs in response to different
environmental conditions (Veron 1995). (iii). Hybridization and reticulate evolution:
Mass spawning produces opportunities for hybridization between species because many
corals spawn simultaneously. Some corals are long-lived at the colony level (hundreds
of years or more), and geographically widespread. Changes in ocean circulation may
introduce genetically and morphologically disparate populations, or create opportunities
for hybridization between species vis-à-vis Veron's (1995) theory of reticulate evolution
by sea surface vicariance.
The goal of this study is to characterize the genetic relationships between P.
lobata populations collected across a wide geographic range (Panamá, the Galápagos,
Easter Island, Tahiti, Rarotonga, Fiji, and Australia’s Great Barrier Reef). Genetic
relationships were examined with a molecular analysis of variance (AMOVA), which
allows the rejection of the null hypothesis of panmixia, by simulating a null distribution
by permutation of haplotypes (Excoffier, et al. 1992; Weir, 1996). The approach
calculates statistics that partition the covariance between and within groups. The
relationships were further examined with a Nested Clade Analysis (NCA), which
explicitly tests the null hypothesis of no association between geography and phylogenetic
78
structure (Templeton 2001). This approach makes use of information contained in
haplotype networks to distinguish between historical population events (such as
fragmentation, long distance colonization, or range expansion), and population structure
(such as low recurrent gene flow due to isolation by distance). The approach has the
additional advantage that the inferences are guided by explicit and objective criteria that
can also indicate a lack of statistical power from inadequate sampling.
A secondary goal of this study is to determine the morphometric relationships
between regions, and to determine if they are similar to the genetic relationships.
Previously (Chapter II), it was discovered that the individuals from Panamá are
genetically distinct from the specimens collected from all other regions. A principal
component discriminant analysis of skeletal measurements was employed to determine if
the Panamá specimens are morphometrically distinct, and to examine the morphometric
relationships between geographic regions. The genetic and morphological relationships
between geographic regions were then examined in the context of satellite data on
prevailing ocean surface currents.
METHODS Genetic analysis
Small, fragments, ca. 10-15 grams of tissue and skeleton were removed from colony
edges, or protuberances. In Rarotonga and Australia populations, small tissue samples
were collected without skeletal vouchers. Samples were collected at least 10 meters
apart to avoid collecting colonies that originated from clonal propagation or
79
fragmentation. Samples were preserved in 95-100% ethanol. The samples were
divided into several pieces when returned the laboratory, a small piece was stored in fresh
ethanol at -20°C for genetic analysis, and larger pieces were placed in bleach to dissolve
the soft tissue, prior to drying. Voucher specimens, and scaled digital microscope
images were collected for the majority of specimens and are available upon request.
Table III-1 summarizes the geographic location of the samples collected, the collector
and the date of collection. DNA extraction, PCR, cloning and sequencing are described
in detail in Chapter II. Each sequence of the entire ITS region (ITS-1, 5.8S, ITS-2), was
sequenced in two directions, which allowed for complimentary strand conformation of
the accuracy of each sequence. At least three individuals were genetically sampled from
Panamá, Galápagos, Easter Island, Tahiti, and Fiji. At least 3 molecular clones were
sequenced from most colonies. Sequence alignment was performed in ClustalW
(Thompson et al. 1994) a gap opening penalty [GOP] of 2, and a gap extension penalty
[GEP] of 1, was selected (see Chapter II for more details on sequence alignment).
There were few alignment gaps, and very few positions were ambiguous.
A cladogram was constructed for all 64 sequences using the Neighbor-Joining (Saitou
and Nei 1987) method Figure III-1. Genetic distances were calculated using Kimura's
(1980) two-parameter model. The tree was bootstrapped 1000 replicates, implemented
in MEGA 2.1 (Kumar et al. 2001). The cladogram is intended to indicate relative
similarity between sequences, and not intended as a phylogeny; many of the assumptions
of phylogenetic methods are violated by population level processes. Similarly,
Maximum Likelihood and Parsimony methods implemented in PHYLIP version 3.6
80
(Felsenstein 2002), and MEGA 2.1 (Kumar et al. 2001) yielded no clear and consistent
population level relationships. Sequences were grouped according to region, and
average distance within and between regions was calculated separately for the ITS-1 and
ITS-2 region (Table III-2) in MEGA 2.0 (Kumar et al. 2001).
A molecular analysis of variance (AMOVA) was conducted in Arliquin v 2.0
(Schneider et al. 2000), with a transition transversion weight of 2:1 and a gap weight of
one (alternative weighing schemes did not significantly alter the outcome). Distances
were calculated with the Kimura (1980) model, and a 0.2 gamma shape parameter (the
shape parameter was estimated in PHYML v 1.0 (Guindon and Gascuel 2002), by the
maximum likelihood method implemented in the program. A separate AMOVA was
performed on the entire data set (including all molecular clones), and then on separate
subsets of one molecular clone per individual, in order to determine if the analysis was
sensitive to differences in sample size between populations. Each subset reflected
highly significant genetic structure between geographic regions (Table III-4).
Significance tests were carried out with 1000 permutations to generate a null distribution
under the assumption of no genetic structure (Excoffier, et al. 1992; Weir, 1996). Fst
statistics were calculated in Arliquin v 2.0 (Schneider et al. 2000), and tested for
significance by 1000 permutations.
A nested clade analysis (NCA) was performed on a subset of the sequences following
the methods outlined in Templeton et al. (1987), and Templeton and Sing (1993). A
haplotype network of the 95% most probable connections was estimated using the
statistical parsimony method, implemented in the computer program TCS v.1.13
81
(Clement et al. 2000). The entire ITS region, and the ITS-1 region alone, contained a
large number of complex reticulations in the haplotype network. Analysis of the ITS-2
resulted in a single network, with no reticulations, which suggests that the levels of
recombination and homoplasy for this data set are relatively low and suitable for the
NCA procedure. Several nearly identical sequences from the same individual have the
potential to severely bias the NCA procedure; therefore, haplotypes from the same
individual that were identical or nearly identical (separated by less than 3 substitutions)
were excluded from the analysis. This resulted in several individual specimens that
contained two disparate haplotypes. The disparate haplotypes did not cluster together,
and therefore should not bias the analysis. The intragenomic variability was quite low,
and many specimens were represented by a single haplotype (see Table III-2 and Figure
III-1). The haplotype network was nested according to rules described in Templeton et
al. (1987), and Templeton and Sing (1993); The procedure joins haplotypes separated by
one mutational event into 1-step clades proceeding from the exterior to the interior of the
network, all 1-step clades separated by one mutational event are then joined into 2-step
clades, and so on, until the entire cladogram is nested in to a single clade (see Figure III-
3). NCA was performed in Geodis v 2.0 (Posada et al. 2000), and the results were
interpreted from the inference key provided with the program.
The geographic distances entered in Geodis, were in the form of a distance matrix of
all possible pairwise distances (in kilometers) between regions, which was estimated by
finding latitude and longitude (accurate only to degrees and minutes) from the Getty
Thesaurus of geographical names online; http://www.getty.edu/. The distances in
82
kilometers between each population were estimated by calculating the great circle
distance between pairs of latitude/longitude co-ordinates using the Lat-Long Converter at
http://www.wcrl.ars.usda.gov/cec/java/lat-long.htm. The GeoDis program calculates
exact permutational contingency tests on two statistics Dc and Dn at each hierarchical
nested level. Dc is a measure of the geographic distribution of a clade, and Dn measures
how widespread a nested clade is relative to other clades in the same nested category,
distances between interior and tip clades (I-T) distances are also calculated (Posada et al.
2000, Templeton et al. 2001). An inference key is provided with the program, with
explicit criteria that allow objective, consistent interpretations of the results, based on
expectations of coalescent theory and computer simulations (Posada et al. 2000,
Templeton et al. 2001). The inference key can also indicate if the sampling design or
the sample size is inadequate to draw conclusions.
Morphometric analysis
For each skeletal voucher, at least 3 Digital images were captured at 18X
magnification using a dissecting microscope attached to a digital CCD videocamera, and
a digital frame-capturing device (ATI all-in-wonder card, ATI technologies Inc.). A
monofilament line of known thickness (0.16mm) was used as a reference for scaling each
image. The images were scaled, and measured using the program Scion Image (Scion
Corporation 2000). Ten corallites for each individual with a voucher specimen (listed in
Table III-1) were measured. The definitions of taxonomic characters are based on Veron
2000, Weil 1992, Weil et al. 1992)
83
For each corallite, a series of 29 X-Y point coordinates were digitized according to
prominent skeletal landmarks related to septal length and relative position depicted in
Figure III-5. The distance between any of the landmark coordinates could then be
calculated. For each corallite, the following traits were measured; 41 linear
measurements between selected point coordinates (Table III-5 and Figure III-5), 2 area
measurements (fossa and corallite area), and 3 discrete variables; number of Pali, number
of radi, and ventral triplet margins fused, free, or tridented. Nine measurements were
proportions of several linear measurements, and four were averages.
A forward stepwise discriminate analysis was implemented in Systat v.9 1998 (SPSS
inc.) All variables in Table III-5 were selected initially, and automatic forward stepping
with default options was selected. The aim of the discriminate analysis is to find a
linear combination of morphometric measurements that best discriminates between user-
defined groups.
In order to examine the relationship between morphology and genetic distance
between populations, distance matrices of averaged genetic distance and average
morphological distance were compared using the Mantel test, implemented in Arliquin v
2.0 (Schneider et al. 2000). The significance tests of linear regressions of distance
matrices are not reliable due to violations of assumptions of independence between data-
points. The Mantel test allows for autocorrelation within a matrix and tests for
significant correlations between matrices by a permutation procedure (Mantel, 1967;
Smouse et al. 1986).
84
RESULTS
Table III-1 indicates the sample size, collector, date and nucleic acid properties of the
ITS region. Table III-2 indicates the pairwise average genetic distances between
populations. The four individual specimens collected from Panamá, originally
identified as Porites lobata were genetically distinct from the 19 specimens collected
across a wide geographic range (Galápagos, Easter Island, Tahiti, Fiji, Rarotonga and
Australia). The Panamá specimens are reciprocally monophyletic under distance,
parsimony and likelihood methods (Chapter II), and will hereafter be referred to as P.
lobata-panama. The maximum difference between the 57 sequences collected from P.
lobata individuals across a broad geographic range differed by only 1.85%. The seven
P. lobata-panama sequences differed from P. lobata by between 6.03% and 6.63%.
The ITS-2 on average differed more between P. lobata populations than the ITS-1;
however, the ITS-1 was more variable between species (Table III-2, and Figure III-2).
Figure III-2 is a Neighbor-Joining distance cladogram that graphically illustrates the
average differences between geographic regions in the ITS-1 and ITS-2. Most regions
have similar branch lengths; however, Easter Island has longer branch lengths when the
entire ITS region, or only the ITS-2 is considered. Fiji has longer branch lengths when
only the ITS-1 is considered (Figure III-2B). Figure III-1 is a Neighbor-Joining
cladogram between all molecular clones used in this study (intended to show distance
relationships, not as a phylogeny). The majority of molecular clones from the same
specimen were very similar, and clustering most often occurred between sequences from
the same individual. Distance, maximum likelihood and parsimony methods are
85
inconsistent on the proximal placement of Easter Island individuals, and no clear and
consistent geographic patterns are readily apparent (Figure III-1).
In order to determine if any geographic structure between the populations is present, a
molecular analysis of variance (AMOVA) was performed (Excoffier et al. 1992; Weir,
1996). The AMOVA indicates that differences between geographic regions are highly
significant (Table III-4). When all sequences are included, nearly 20% of the variance
is attributed to differences between geographic regions (p <0.0001), and 80% is due to
variance within populations. Highly significant geographic structure between
geographic regions was also evident when one sequence was chosen per individual
(repeated multiple times with alternative molecular clones), or when all Easter Island
sequences were excluded from the analysis (Table III-4). This indicates that the
significant geographic structure is not solely due to the most genetically distant group
(Easter Island), or to problems associated with sampling unequal numbers of sequences
per individual per region. The calculation of Fst statistics confirms the indication by the
averaged distance data that Easter Island is the most genetically distinct geographic
region. Easter Island has the highest and most significant differences according to the
permutation analysis (Table III-3 B).
Phylogeographic Nested clade analysis (Figure III-3 and Table III-4) indicates
that the null hypothesis of no association between phylogenetic structure and geographic
structure can be rejected for two clades; Clade 3-1 and Clade 3-3. An exact contingency
tests of the next highest clade, is highly significant for clade 4-1 (of which 3-1 is a
member); p < 0.01, and clade 4-2 is significant (of which 3-3 is a member); p < 0.05 (see
86
Table III-4). The inference key for NCA (Templeton 1998, Posada et al. 2000)
indicates that the expectations of restricted recurrent gene flow and isolation by distance
are met for both clade 3-1 and 3-3. All sequences from the most geographically isolated
population (Easter Island) are contained in the two significant clades. Clade 3-1
contains sequences from Easter Island and Rarotonga, and Clade 3-3 contains individuals
from Easter Island and the Galápagos. Rarotonga and the Galápagos are the two most
likely candidate source populations for Easter Island, as implied by the ocean surface
current vectors generated from satellite data (Figure III-4 B & C). The satellite data also
indicates a strong unidirectional current flowing west and slightly southwest from the
Galápagos to the south central populations. The current vectors connect the populations
in a way that appears similar to the averaged genetic similarity between populations.
The haplotype network (Figure III-3) indicates that haplotypes are most frequently shared
between the Galápagos and Tahiti followed by Rarotonga.
Morphometric analysis
Corallites generally appear to vary by region, as illustrated in Figure III-6. The
majority of traits measured (Table III-5) exhibited significant differences among
geographic regions. According to a model one ANOVA, and paired comparisons with
Tukey's HSD correction, nearly all measurements showed some significant differences
between regions (Panamá, followed by Easter Island were distinct most frequently).
Examples of some of the significant differences are illustrated in Figure III-7. A
stepwise canonical discriminant analysis, that initially included all the measured variables
87
in Table III-5, indicated that the Panamá individuals were distinguishable from all other
geographic regions; Wilks' lambda = 0.076, p < 0.0001 (Figure III-8). The variables
TRI, NP, SW/L, 23:3/L, PA, and CA had the largest influence on discriminating between
populations. The Jackknifed classification matrix indicates how many corallites were
correctly classified by groupings based on regions, 95% of Panamá region corallites were
classified correctly. The eigenvalues indicate that the first two factors account for the
largest portion of the variance. Neighboring populations, overlap more than populations
at extreme ends of the geographic range (for example, Galápagos and Fiji are nearly
completely non-overlapping).
Linear regressions of average genetic distance between populations with average
morphologic distance between populations were significant for 37 of the 58 variables
measured (64%). Significance values of linear regressions of distance matrices are not
reliable because of violated assumptions of independence; however, the more
conservative Mantel test indicated that 12 of the 58 morphometric variables (21%), were
significantly related to the average genetic distance between regions (Figure III-9).
DISCUSSION
Panmixia is generally assumed in geographically widespread species, especially those
that have long planktonic larval durations (surviving for weeks or more), because ocean
currents are capable of dispersing propagules over enormous distances (Faldallah 1983).
The detection of isolation by distance, in one of the most geographically widespread coral
species suggests that the paradigm of Panmixia is an oversimplification. If gene flow is
88
directly mediated by prevailing ocean currents (which can be strongly unidirectional),
then simple models of isolation by distance are inadequate. Several observations in this
study suggest that an isolation by ocean currents model would fit the data more
accurately than isolation by distance. Easter Island shares haplotypes with the two
populations that are in the direct path of the current vectors in Figure III-4 (Rarotonga
and the Galápagos). The Galápagos is located in the middle of the very strong south
equatorial current, the current vectors flow in order of strength to Tahiti, Rarotonga, Fiji
and Australia (Figure III-4 B & C). According to the haplotype network, Tahiti most
substantially overlaps with the Galápagos haplotypes, followed by Rarotonga and Fiji
(Figure III-3). The average genetic distance between populations reflects a similar
pattern (Figure III-3, and Figure III-2). If we assume that gene flow is mediated by
prevailing currents, then the Galápagos is likely to be an important source population, or
at least a critical stepping stone in the convoluted path of global ocean circulation. P.
lobata is one of the few dominant corals of the Galápagos archipelago, and the only
species of Porites that occurs there (Glynn and Wellington 1983). The fauna is
depauperate with very little reef formation. It is also located at the geographic center of
the El Nino Southern Oscillation, which causes water temperatures to rise and is
associated with mass coral bleaching and mortality. The current vectors in Figure III-4
do not readily suggest a plausible candidate source population for the Galápagos.
Future studies in the Eastern Pacific and the Northern Hemisphere, should address this
issue.
89
The phylogeographic NCA analysis rejects the null hypothesis of no association
between phylogenetic and geographic structure. Under the null hypothesis of panmixia,
all clades have the same geographic center, permutations of the data to generate a null
distribution, allowing for significance testing against random fluctuations expected from
genetic drift, or sample error (Templeton 2001). The technique has explicit criteria for
distinguishing between historical and population level processes, based on expectations
from simulations and coalescent theory (Templeton 1998, 2001). One of the predictions
of coelecent theory is that older haplotypes will be more common in a given sample, and
that interior clades (with multiple connections) are generally younger than tip clades.
The Easter Island haplotypes all occurred in tip clades, and the clade distance (Dc) and
the nested distances were significantly small, while the Dc of the interior clades, and the
I-T distances were significantly large, which are predicted by isolation by distance. The
inference of isolation by distance is strengthened by the fact that for the geographically
restricted clades, the union of the ranges roughly corresponds to the range of the interior
clades (i.e. clade 2-2 and 2-7) within the same nested group, which is predicted by the
inference key (Templeton 2001).
The haplotype network (Figure III-3) appears to have symmetrical properties, and
many of the specimens sampled contain two distinct haplotypes indicating that there may
be two distinct haplotype families that have undergone a parallel history. Two active
arrays of ribosomal genes (nucleolus organizer regions) located on separate
chromosomes, or a gene duplication event could explain this pattern. Moderately
divergent intragenomic paralogues have been associated with slower rates of crossover
90
and gene conversion between separate chromosomal lineages (Arnheim et al. 1980,
Polanco et al. 2000). Alternatively, hybridization and introgression between two coral
species, or incomplete lineage sorting could be invoked as explanations. It is unlikely
that one of the haplotype families (Clade 4-1 or 4-2 in Figure III-3) is a non-functioning
pseudogene. Pseudogenes have been discovered in Acropora species; however, they are
usually associated with substantial levels of intragenomic variation. (Odorico and
Miller 1997; van Oppen 2000). Our survey of other Porites and Siderastrea species
(Chapter II), suggests that levels of intragenomic variability in Porites and Siderastrea
are very low, and relatively low variability have been observed in most Scleractinian
species surveyed to date (reviewed in Marquez et al. in press). We favor the existence
of two nucleolus organizer regions, or hybridization between a species that has not yet
been sampled as the most likely hypotheses.
Inragenomic variation in the ITS region was substantially lower than reported
previously by Hunter et al (1997) in P. lobata from Hawai'i (6%). Hawai’i is an
isolated archipelago with several endemic species of Porites, where an adaptive radiation
may have occurred. It is possible that a cryptic species within the P. lobata complex
was sampled, or that the sequence (AF180115) contains noise, due to absence of
complementary strand confirmation. Mutations appear scattered throughout the
sequence in a manner similar to sequences with high noise or low signal. In our data
set, differences between most Porites species tend to be constrained to several specific
regions of the sequence (Chapter II). Further studies of the Hawai’ian Porites fauna
91
will undoubtedly resolve this issue, as well as clarify the relationships between the many
unique growth forms endemic to the region.
The morphological differences in corallite level characteristics, and differences in
gross colony morphology are consistent with the genetic isolation by distance and low
recurrent gene flow predicted by the nested clade analysis. Many Easter Island P.
lobata colonies are strikingly different in colony appearance from all other geographic
regions, forming tall columnar fins or peaks. The distinctiveness of the population, in
terms of corallite and colony level characteristics, as well as genetic differences, might be
expected as it is located thousands of kilometers away from neighboring populations, and
it is located in the middle of the counter clockwise flowing South Pacific Gyre.
Although Easter Island is the most genetically distinct population (Table III-2), the
genetic differences are on a scale consistent with intraspecies variation (1.5-1.8%). The
nested clade analysis includes explicit criteria that are capable of detecting historical
patterns such as past fragmentation, or range expansion (including long distance
colonization) (Templeton 1998, 2001). The Easter Island clades met the criteria of
recurrent low levels of gene flow. The correlations between morphology and genetics,
and the pattern of morphometric isolation by distance (Figure III-8) lend further support
to this hypothesis.
The reciprocally monophyletic genetic differences between P. lobata and P.
lobata-panama are as high as differences between other Porites species (Chapter II), and
the morphometric data confirm this result. It is possible that both P. lobata-panama and
P. lobata are present in Panamá, and that only P. lobata-panama was sampled due to
92
patchy or habitat specific occurrence, or difference in abundance. Likewise, the small
sample sizes in each region have the potential to exaggerate some differences and
therefore bias the overall result. Nevertheless, each individual was considerably more
similar to other individuals from the same region (Figure III-8) and there was strong
concordance between the molecular and morphometric data that is unlikely to be the
result of chance or sampling artifact.
The overall pattern is one of gradual changes in genetic and morphological
variation between geographical regions. The variation generally increases with
distance, unless overridden by prevailing ocean surface currents. The patterns are best
explained by selection operating on a regional scale, with the cohesive forces of low to
intermediate levels of recurrent gene flow mediated by ocean currents. Although the
samples in this study span more than ten thousand kilometers, only a sparse sampling of a
small portion of P. lobata's geographic range is represented. This study is therefore
only a preliminary view of the complex history of P. lobata in space and time.
93
TABLES
Table III-1
Length variation, percent G + C content, number of individuals, number of sequences, geographic
region, collector and date for the ITS-1 and ITS-2 sequences collected for this study. Abbreviations are as
follows: EP, Pacific; CP, Central Pacific WP, Western Pacific; * P. lobata-panama was collected from Uva
and Saboga Panamá. Collectors and dates are represented by numbers in superscript; 1 = G. Wellington
(99), 2 = M. Takabayashi (98), 3 = Z. Forsman (98). Samples in bold letters indicate that a skeletal
voucher specimen was collected. The 5.8S gene had few polymorphisms, and a nearly constant length of
106-107nt, and a 51% G+C content
94
ITS-1 ITS-2 Length Length No of No of Species Region (bp) %(G+C) SE (bp) %(G+C) SE Individuals Sequences P. lobata-panama* Panama* (EP)1 303-311 43.27 0.19 228-229 44.91 0.23 4 7 P. lobata Easter Isl. (CP)1 303-312 42.02 0.47 215-226 43.89 0.22 4 10 " " Australia (WP)2 305-325 42.17 0.43 210-223 43.91 0.75 2 7 " " Rarotonga (WP)1 306-309 42.27 0.26 207-226 44.16 0.45 3 9 " " Tahiti (CP)1 306-309 41.96 0.43 207-231 43.89 0.43 3 9 " " Galapagos (EP )3 306-307 42.29 0.34 209-225 44.04 0.49 4 15 " " Fiji (CP)1 305-309 41.91 0.41 215-223 43.81 0.83 3 7 Total 23 64
Table III-1
95
Table III-2
Matrix of genetic distance between populations (in substitutions per site,
calculated by the Kimura 1980 method), and standard errors. Numbers in bold script
along the diagonal represent intra-population means. Standard errors are in italic script.
Distances were calculated separately for the ITS-1, and ITS-2. Mean differences
between populations, and net distance between populations are shown. Standard errors
were estimated by 1000 bootstrap replicates implemented in MEGA 2.1 (Kumar et al.
2001). Abbreviations are as follows: A = Australia, E = Easter Isl., F = Fiji, G =
Galápagos, R = Rarotonga, T = Tahiti, P = Panamá.
96
Mean Difference Within and Between Groups Net Difference Between Groups ITS-1 ITS-1
E 0.013 0.010 ±0.004 0.006 0.005 0.004 0.005 0.010 E 0.002 ~ 0.003 0.002 0.002 0.002 0.010
F 0.018 0.019 0.016 ±0.006 0.005 0.005 0.005 0.010 F 0.004 0.006 ~ 0.001 0.002 0.002 0.009
G 0.014 0.014 0.015 0.011 ±0.004 0.004 0.004 0.010 G 0.003 0.004 0.001 ~ 0.001 0.001 0.009
R 0.013 0.012 0.016 0.012 0.009 ±0.004 0.004 0.010 R 0.003 0.003 0.004 0.002 ~ 0.001 0.010
T 0.014 0.013 0.016 0.012 0.012 0.010 ±0.004 0.010 T 0.003 0.003 0.003 0.002 0.002 ~ 0.009
P 0.027 0.033 0.034 0.030 0.033 0.030 0.003 ±0.002 P 0.020 0.027 0.025 0.023 0.027 0.024 ~
ITS-2 ITS-2 A E F G R T P A E F G R T P
A 0.019 ±0.006 0.007 0.004 0.004 0.005 0.004 0.019 A ~ 0.004 0.001 0.001 0.002 0.001 0.019
E 0.029 0.019 ±0.005 0.006 0.006 0.006 0.006 0.019 E 0.011 ~ 0.004 0.002 0.002 0.004 0.018
F 0.016 0.024 0.009 ±0.003 0.004 0.004 0.003 0.018 F 0.002 0.011 ~ 0.001 0.001 0.001 0.019
G 0.020 0.023 0.014 0.016 ±0.004 0.004 0.003 0.019 G 0.002 0.006 0.002 ~ 0.001 0.001 0.018
R 0.019 0.022 0.016 0.018 0.015 ±0.005 0.004 0.019 R 0.002 0.005 0.004 0.003 ~ 0.001 0.019
T 0.014 0.024 0.010 0.013 0.015 0.009 ±0.003 0.019 T 0.001 0.011 0.002 0.001 0.003 ~ 0.019
P 0.110 0.114 0.106 0.108 0.117 0.108 0.005 ±0.002 P 0.110 0.114 0.110 0.110 0.120 0.114 ~
Table III-2
97
Table III-3
AMOVA tables of genetic structure within and between geographic regions. (A).
All sequences included. (B). Table of Pairwise Fst values (below diagonal), and
pairwise significance values (above diagonal), were calculated using Arliquin v 2.0
(Schneider et al. 2000). Significance values were obtained by 1023 permutations of the
data to generate a null distribution. (C). An example of one of the subsets of sequence
per individual (this was repeated several times with alternative sequences/individuals
from a region) -the genetic structure was still highly significant, indicating that the
genetic structure is not an artifact of the sampling method. (D). Significant
differences between regions still occur when all Easter Island sequences were excluded
from the analysis. This indicates that the genetic structure is not solely due to Easter
Island (the most genetically distinct population).
98
E A R T G F E ~ 0.0001 0.0001 0.0001 0.0001 0.0001 A 0.34 ~ 0.06 0.07 0.01 0.15 R 0.32 0.12 ~ 0.01 0.001 0.01 T 0.36 0.09 0.15 ~ 0.05 0.01 G 0.25 0.12 0.16 0.07 ~ 0.05 F 0.38 0.07 0.20 0.15 0.09 ~
Source of Variation d.f. S.S. V.C. % Variation Among Regions 5 36.545 (a) 0.72 12.43 p < 0.02 Within Regions 13 65.75 (b) 5.06 87.57 Total 18 102.296 5.78
Source of Variation d.f. S.S. V.C. % Variation Among Regions 4 29.38 (a) 0.44 11.78 p < 0.0001Within Regions 42 138.64 (b) 3.30 88.22 Total 46 168.02 3.72
Source of Variation d.f. S.S. V.C. % Variation Among Regions 5 59.115 (a) 0.89 19.79 p < 0.0001 Within Regions 50 181.076 (b) 3.62 80.21 Total 55 563.83 10.6
Table III-3
(A) All Sequences Included
(B) Pairwise Fst and significance values between geographic regions
(C) One sequence per individual
(D) Easter Island excluded
99
Table III-4
The results of the nested clade analysis. Clades in gray are interior, others are tip
clades. Significantly large values are denoted by L, small values by S, the level of
significance is indicated as follows: * = p < 0.05 ** = p < 0.01, Dc = Clade distance,
Nc = Nested clade distance. I-T distances are indicated for significant clades only.
The inference chain (according to the inference key of Templeton 1998, and Posada et al.
2000) is indicated underneath the significant clade, IBD is an abbreviation for Isolation
By Distance.
100
Haplotypes 1-step 2-step 3-step 4-step Name Dc Dn Name Dc Dn Name Dc Dn Name Dc Dn Name Dc Dn r1-2 1-1 0 3412 r6-4 0 0
LITERATURE CITED Arnheim, N. M., Krystal, M., Shmickel, R., Wilson, G., Ryder, O. and Zimmer, E. (1980). Molecular evidence for genetic exchanges among ribosomal genes on nonhomologous chromosomes in man and apes. Proc. Natl. Acad. Sci. USA 77, 7323-7327. Bernard, H. M. (1902). The species problem in corals. Nature 65, 560. Bonjean, F. and Lagerloef, G. S. E. (2002). Diagnostic model and analysis of the surface currents in the tropical Pacific Ocean. Journal of Physical Oceanography 32, 2938-2954. Clement, M., Posada, D. and Crandall, K. A. (2000). TCS: a computer program to estimate gene genealogies. Molecular Ecology 9, 1657-1659. Dana, J. D. (1846). Zoophytes, pp. 740: United States exploring Expedition. Excoffier, L., Smous, P. and Quattro, J. (1992). Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data. Genetics 136, 343-59. Fadlallah, Y. H. (1983). Sexual reproduction, development and larval biology in scleractinian corals: a review. Coral Reefs, 129-150. Felsenstein, J. (2002). Phylogeny Inference Package (PHYLIP) Version 3.6, Univ. of Washington, Seattle. Frost, S. H. (1977). Miocene and Holocene evolution of Caribbean province reef-building corals. Proc. Third Int. Coral Reef Symp., Miami 2, 353-359. Glynn, P. W. W., G.M. (1983). Corals and coral reefs of the Galapagos Islands, pp. 330. Berkley: Univ. of California Press. Guindon, S. and Gascuel, O. (2002). PHYML; a simple, fast and accurate algorithm to estimate large phylogenies by maximum liklihood. Hunter, C. L., Morden, C. W. and Smith, C. M. (1997). The utility of ITS sequences in assessing relationships among zooxanthellae and corals. Proc. 8th int coral reef sym. , 1599-1602.
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Kimura, M. (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111-120. Knowlton, N. (1993). Sibling species in the sea. Annu. Rev. Ecol. Syst 24, 189-216. Kumar, S., Tamura, K., Jakobsen, I. and Nei, M. (2001). MEGA2: Molecular Evolutionary Genetics Analysis software Version 2.1. Tempe Arizona: Arizona State University. Link, H. F. (1807). Bescheibung der Naturalein. Sammlungen der Universaitat Rostock, 3, 161-165. Mantel, N. (1967). The detection of disease clustering and a generalized regression approach. Cancer Research 27, 209-220. Marquez, L., Miller, D., MacKenzie, J. and van Oppen, M. J. H. (in press). Psudogenes contribute to the extreme diversity of nuclear ribosomal DNA in the hard coral Acropora. Odorico, D. M. and Miller, D. J. (1997). Variation in the Ribosomal Internal Transcribed Spacers and 5.8S rDNA Among Five Species of Acropora (Cnidaria;Scleractinia): Patterns of Variation Consistent with Reticulate Evolution. Mol. Biol. Evol. 14, 465-473. Polanco, C., Gonzalez, A. I. and Dover, G. A. (2000). Patterns of variation in the intergenic spacers of ribosomal DNA in Drosophila melanogaster support a model for genetic exchanges during X-Y pairing. Genetics 155, 1221-9. Posada, D. (2000). GeoDis: a program for the cladistic nested analysis of the geographical distribution of genetic haplotypes. Molecular Ecology 9, 487-488. Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425. Schneider, S., Roessli, D. and Excoffier, L. (2000). Arlequin ver. 2.000: A software for population genetics data analysis. Switzerland: Genetics and Biometry Laboratory, University of Geneva. Smouse, P. E. and Long, J. E. (1986). Multiple regression and correlation extensions of the Mantel Test of matrix correspondence. Systematic Zoology 35, 627-632. Templeton, A. R. (1998). Nested clade analyses of phylogeographic data: testing hypotheses about gene flow and population history. Mol Ecol 7, 381-97. Templeton, A. R. (2001). Using phylogeographic analysis of gene trees to test species status and processes. Molecular Ecology 10, 779-791.
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Templeton, A. R., Boerwinkle, E. and Sing, C. F. (1987). A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping. I. Basic theory and an analysis of alcohol dehydrogenase activity in Drosophila. Genetics 117, 343-351. Templeton, A. R. and Sing, C. F. (1993). A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping. IV. Nested analysis with cladogram uncertainty and recombination. Genetics 134, 659-669. Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680. van Oppen, M. J. H., Willis, B. L., Van Vugt, H. W. J. A. and Miller, D. J. (2000). Examination of species boundaries in the Acropora cervicornis group (Scleractinia, Cnidaria) using nuclear DNA sequence analyses. Molecular Ecology 9, 1363-1373. Vaughan, T. W. (1907). Recent Madrporaria of the Hawaiian Islands and Lysan. US National Mus Bull 59, 427pp. Veron, J. (1995). Corals in space and time; the biogrography and evolution of the scleractinaia. London: Cornell. Veron, J. E. N. (2000). Corals of the World, vol. 3 (ed. M. Stafford-Smith). Townsville, Australia: Australian Institute of Marine Science. Veron, J. E. N. and Pichon, M. (1982). Scleractinia of Eastern Australia part 4, Family Poritidae. Australian Institute of Marine Science Monograph Series 5, 159. Weil, E. F. (1992). Genetic and morphological variation in Caribbean and eastern Pacific Porites (Anthozoa, Scleractinia), preliminary results. Proc 7th Int. Coral Reef Sym. Guam 643-656. Weil, E. F. (1992). Genetic and morphological variation in Porites (Cnidaria, Anthozoa) across the Isthmus of Panama. In Ph.D. Dissertation, pp. 327. Austin TX: University of Texas. Weir, B. S. (1996). Genetic Data Analysis II: Methods for discrete population genetic data. Sunderland, MA, USA: Sinauer Assoc. Inc.
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IV. The Siderastrea glynni (Scleractinia: Siderastreidae) Paradox: A Critically Endangered Species Or A Stowaway From The Caribbean? ITS Region Sequences Are Shared With S. siderea.
ABSTRACT The extremely rare Panamanian endemic Siderastrea glynni, is one of only two documented critically endangered species of hermatypic coral. S. glynni is the only member of the genus that occurs in the entire eastern Pacific province. Only 5 individuals have been discovered, currently only 4 exist. A comparison of cloned sequences of the Internal Transcribed Spacer (ITS region) reveals that all four S. glynni individuals have extremely low nucleotide diversity, with 15 molecular clones from 4 individuals differing by only 0.17%, -designated as the Clade A haplotype. All individual S.siderea collected from the Caribbean side of Panamá contain the exact same haplotype in roughly equal proportions to an additional haplotype; the Clade B haplotype. The average nucleotide distance between S. glynni and S. siderea is only 0.83%, whereas the maximum distance between the A and B haplotypes is 1.7%. The widely accepted view that S. glynni originated from dispersal from the western Pacific is highly unlikely. There are two remaining alternative hypotheses: (1) A geminate relationship between S. glynni and S. siderea. (2) a more recent colonization through the isthmus of Panamá. Previously published mutation rates of the ITS region were examined in order to determine if the maximum distance between S. glynni and S. siderea haplotypes are consistent with a 3.5 million year divergence. The mutation rates vary by only 5 fold between a wide variety of organisms. The mutation rates cannot reject Hypothesis (1), however this is only if the mutation rate is unusually slow (0.2%), and if the assumptions of a molecular clock are not sufficiently violated. Phylogeographic Nested Clade Analysis was used to make inferences about the information contained in a haplotype network generated by statistical parsimony. The analysis supported the inference of long distance colonization, which is more consistent with a contemporary relationship between S. glynni and S. siderea. Although neither hypothesis can be ruled out, either hypothesis has important implications about population level processes that govern the tandem arrays of ribosomal genes and spacers, such as lineage sorting, genetic drift, founder effect, and concerted evolution. Further study is necessary to solve the S. glynni paradox.
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INTRODUCTION
There are only two documented critically endangered species of coral; S. glynni and
Millepora boschmai. S. glynni, is endemic to Panamá, and extremely rare. Only five
colonies have been discovered, and currently only four exist (Budd and Guzman 1994).
Budd and Guzman (1994) hypothesized that S. glynni may have originated from a small
founding population from the central Pacific, perhaps from a single rare dispersal event.
The hypothesis is based on the rationale that S. glynni is morphometrically distinct from
the Caribbean fauna, and most similar to the central Pacific species S. savignyana. The
alternative hypothesis is that a geminate species may exist in the Caribbean, and S. glynni
is the last relict of a population that was fragmented by the closure of the Tropical
American Seaway, which occurred approximately ~3-3.5 MYA (Kegwin 1982).
A third seemingly unlikely hypothesis is that S. glynni is a non-indigenous species
that has somehow been transported from the Caribbean. The hypothesis would require
that the soft bodied organism survived prolonged passage through the fresh water of the
Panamá canal, perhaps in floating debris, in ships ballast water, as larval spat that settled
on an anchor chain, or some other such mechanism. All five colonies were discovered
within a few square meters of each other, they were small and similar size, and they were
located downstream from the Pacific opening of the Panamá canal. The observed
differences in morphology (Budd and Guzman 1994) could simply be due to phenotypic
plasticity, arising from environmental differences between the Caribbean and Eastern
Pacific.
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We collected all of the extant members of Sidereastrea that occur around the
Isthmus of Panamá in order to determine the possible origin of S. glynni. Siderastrea is
one of the few Scleractinian genera that occur on both sides of the Isthmus. S. glynni is
the only extant Siderastrea species in the eastern Pacific, and Siderastrea siderea, S.
radians and S. stellata are the only species that occur in the Caribbean (Veron 2000).
To examine the possibility of a geminate relationship, an accurate estimate of the
mutation rate of the molecular marker in question is necessary. We surveyed the
estimated ITS region mutation rates from the literature on a wide variety of organisms.
Martin and Palumbi (1993) established that mutation rates covary with a number of
characteristics that are likely to influence 'nucleotide generation time', such as generation
time, metabolic rate, body size, rate of cellular division, and DNA repair efficiency. It
is important therefore to take nucleotide generation time into account, when estimating
divergence rates because small short-lived organisms generally have much higher
mutation rates then large long-lived ones.
We employed a phylogeographic Nested Clade Analysis (NCA), which explicitly
tests the null hypothesis of no association between geography and phylogenetic structure
(Templeton et al. 1987; Templeton and Sing, 1993). This approach makes use of
information contained in haplotype networks to distinguish between historical population
events (such as fragmentation, long distance colonization, or range expansion), and
population structure (such as low recurrent gene flow and isolation by distance). The
approach has the additional advantage that the inferences are guided by explicit and
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objective criteria that can also indicate lack of statistical power from inadequate
sampling.
METHODS
Very small tissue scrapings (approximately 10-20mg) were collected from each
specimen. S. radians and S. siderea were obtained from Bocas del Toro on the
Caribbean side of Panamá. S. stellata was collected from Pernambuco State Brazil. S.
glynni was originally discovered near Isla Uraba in the Bay of Panamá, near the Pacific
coast; however, after a mass bleaching event during the 1998 El Nino, the colonies were
moved to the Smithsonian Tropical Research Institute (STRI). Extraction of DNA,
PCR, cloning and sequencing are described in detail in Chapter I. Each molecular clone
was sequenced in two directions, which allowed for complementary strand conformation.
S. glynni was processed 2 months before any other samples, therefore the risk of cross
contamination was minimal. Sequence alignment was performed by hand in Bioedit
(Hall 1999). There were few alignment gaps and no ambiguities. All genetic distances
were calculated with the Kimura (1980) method in Mega 2.1 (Kumar et al. 2001)
A plausible range of mutation rates for the ITS region was determined by examining
the relationship between previously published rate estimates for a wide range of
organisms, compared to an approximation of organismal generation time (Figure IV,
Table IV-3). Tajima's (1993) equal rates test was performed in Mega 2.1. The test
detects significant deviations from expectations of a linear relationship between mutation
rate and time, between an outgroup and two other sequences. The Neighbor-Joining
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cladogram was calculated in Mega 2.1, using 1000 bootstrap replicates, it is intended to
show relative distances between sequences not as a phylogeny.
A phylogeographic Nested Clade Analysis (NCA) was performed following the
methods outlined in Templeton et al. (1987), and Templeton and Sing (1993). A
haplotype network of the 95% most probable connections was estimated using the
computer program TCS v.1.13 (Clement et al. 2000). The network had no reticulations,
which indicates that homoplasy and recombination are relatively low. The haplotype
network was nested according to rules described in Templeton et al. (1987), and
Templeton and Sing (1993); the procedure joins haplotypes separated by one mutational
event into 1-step clades proceeding from the exterior to the interior of the network, all 1-
step clades separated by one mutational event are then joined into 2-step clades, and so
on, until the entire cladogram is nested in to a single clade (see Figure IV-3). NCA was
performed in Geodis v 2.0 (Posada et al. 2000), and the results were interpreted from the
inference key provided with the program.
The geographic distances entered in Geodis, were in the form of a categorical
distance matrix: There were only two populations sampled and there were multiple
sequences sampled per individual; therefore, categorical distances were assigned instead
of geographic distances. Either categorical or continuous variables are allowed in the
analysis (Templeton 1998). Each individual specimen was treated as a separate group,
specimens from the same population were assigned an arbitrary 'close' distance of 1. An
arbitrary 'far' distance of 5 was assigned to individuals separated by the Isthmus of
Panamá. The results of the analysis were identical if alternate distances were assigned,
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as long as the interpopulation distances were larger than intrapopulation distance, and
each category was homogeneous.
The GeoDis program calculates exact permutational contingency tests on two
statistics Dc and Dn at each hierarchical nested level. Dc is a measure of the geographic
distribution of a clade, and Dn measures how widespread a nested clade is relative to
other clades in the same nested category, distances between interior and tip clades (I-T)
are also calculated (Posada et al. 2000, Templeton et al. 2001). Significance testing is
based on simulated null distributions under the expectations of panmixia. The null
distributions are generated by permutations of the data, therefore stochastic variation and
the sample sizes per locality are accounted for.
RESULTS
Thirty-eight contiguous sequences for the complete ITS-1, ITS-2 and 5.8S were
assembled, with at least 3 molecular clones for each Siderastrea species (Table IV-1).
The 5.8S rRNA gene was invariant between all sequences. The A-T content, variations
in length and sample sizes are listed in Table IV-1. Table IV-2 is a distance matrix of
all pairwise comparisons between sequences in this study. All four S. glynni individuals
had remarkably low sequence diversity (0.17%, n=16). Surprisingly, the exact same
sequence was present in both S. glynni and S. siderea individuals. All S. siderea
individuals shared at least one identical sequence with S. glynni (designated as clade A).
All S. siderea individuals contained a second sequence, (designated as clade B) which
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was not present in any of the S. glynni individuals. In other words, all of the S. glynni
haplotypes were nested within the range of S. siderea haplotypes (Figure IV-2).
To examine whether the divergence between the most distinct haplotypes in the
two populations are consistent with a 3.5 million year division it is necessary to have an
accurate estimate of mutation rate for the ITS region. Previously published rate
estimates indicate a strong correlation between approximate generation time and mutation
rate (Figure IV-1 and Table IV-3), a relationship that was established by Martin and
Palumbi (1993). Small short-lived organisms (Algae and Drosophila) have high
mutation rate, where large, long-lived organisms (Birch and Alder trees and primates),
have low mutation rates. The ITS rates across a wide variety of organisms are
surprisingly similar, varying only approximately 6 fold, in contrast mtDNA RFLP data
varies 25 fold in vertebrates (Martin and Palumbi 1993). It would be reasonable to
assume that the minimum possible generation time for a coral colony is on the order of
several years. Hunter (1988) estimated first reproduction in Porites compressa was
approximately 2 years, therefore a plausible maximum and minimum rate based on the
correlation in Figure IV-1 are around 0.6% and 0.2% per million years respectively.
Based on a fossil calibration point in Porites (Chapter II), we estimate the ITS region
substitution rate at approximately 0.4% per million years.
Tajima's relative rate test failed to reject the null hypothesis of equal rates among
the A and B haplotypes, when either S. stellata or S. radians was selected as an outgroup
(chi-square = 1.29, p = 0.257), therefore we assume that mutation rates are proportional
to time, and may be useful for comparing divergences with known historical events.
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Figure IV-2 indicates a Neighbor-Joined cladogram of the substitution distances between
all sequences in this study. Superimposed in the background of the figure is the timing
of the complete closure of the Isthmus of Panamá, which occurred between 3.5 and 3.8
million years ago (Kegwin 1982). The large shaded rectangle indicates the possible
range of the event if the mutation rate for the ITS region in Siderastrea is between 0.2
and 0.6% per million years. The shaded central portion of the rectangle is centered on the
time of the divergence if the rate is 0.4% per million years. It is only possible for the
distance between clade A and B to be 3.5 million years ago if the lowest published rate of
0.2% is assumed. The hypothesis that the distance between clade A and B is 3.5 million
years appears to be unlikely, but it cannot be entirely ruled out.
The results of the phylogeographic NCA analysis are illustrated in Figure IV, and
Table IV-4. The haplotype network had no ambiguous steps or reticulations. All of
the S. glynni haplotypes were nested within the S. siderea clade (Clade A). The NCA
inference key indicates that a long distance colonization event is likely to have occurred.
The clade distances (Dc) are significantly small (p < 0.01), while the nested clade
distances are significantly large (p < 0.01). Significant reversals between Dc and Dn for
a clade generally indicate long distance dispersal events, especially if no intermediate
populations exist (Templeton 1998). The NCA analysis indicated that clade A had the
highest probability of being an older clade, which is based on the predictions of
coalescent theory that older haplotypes will be more frequent in the population, and will
tend to be located in the interior of a network. A separate haplotype network was
constructed that contained only S. siderea samples (data not shown), which also indicated
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that the A clade has the highest outgroup probability. The large numbers of shared
identical sequences, and the NCA inference of long distance colonization suggests a
contemporary relationship between S. glynni and S. siderea.
DISCUSSION
According to our data, the hypothesis that S. glynni originated from dispersal from the
central Pacific can be rejected. Due to the low nucleotide diversity among all four S.
glynni individuals, it is obvious that S. glynni has passed through a population bottleneck;
however, it cannot be determined with certainty if the bottleneck occurred 3.5 million
years ago, or in the last 100 years. Through phylogeographic nested clade analysis, we
can determine that the observed patterns are more consistent with the expectations of a
colonization event by long distance dispersal, than with a historical event such as
allopatric fragmentation. According to the NCA inference key, past fragmentation
events should result at least partially non-overlapping clade ranges, with larger than
average number of intermediate haplotypes (Templeton 1998). The ITS data clearly
nests all of the S. glynni haplotypes as a subset well within the range of the S. siderea
haplotypes. The analysis also indicates that clade A is likely to be older than clade B,
due to its position in the interior of haplotype networks, and its higher frequency in both
populations.
If the two populations were separated for multiple generations, then mutations should
occur independently causing them to become differentiated. It could be argued that
concerted evolution, and high inbreeding in a small population could lead to the fixation
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and preservation of the most commonly occurring ancestral haplotype (in this case the
clade A haplotype); however, it seems unlikely that stochastic processes such as genetic
drift, concerted evolution, and mutation would all cumulatively fail to alter the A
haplotype in either population for generation after generation for 3.5 million years. The
homogenization of ribosomal arrays occurs at a rate that is even faster than the
substitution rate (James et al. 2001), and genetic drift can occur within generations. If
the process of lineage sorting of ancestral alleles is very slow, then it would be expected
that some ancestral alleles could persist in S. glynni over many generations; however,
some alleles completely unique to S. glynni would also be expected. In Porites, a genus
closely related to Siderastrea, the ITS region clearly separated species that were likely to
have diverged between 1.6 and 2.5 million years ago, therefore incomplete lineage
sorting does not necessarily present a problem over these timescales (Chapter II). Out
of all 16 sequences, S. glynni has only five unique mutations, all of which are singleton
mutations occurring only once and scattered throughout the alignment in a manner
consistent with errors associated with Taq polymerase, or base calling errors.
In the context of previously published rate estimates of the ITS region, the
hypothesis that the distance between clade A and B is 3.5 million years cannot be entirely
ruled out; however, nearly all S. glynni sequences are completely identical to S. siderea
sequences, if the difference between 'species' is measured from the least divergent
haplotypes, or the net or average distance between species, then a 3.5 million year
separation time is clearly incompatible with the data.
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From a survey of Porites species (Chapter II), intraspecific nucleotide diversity
in most species is quite similar to that of S. siderea. Similar levels of nucleotide
diversity in multiple species could be related some fundamental properties of concerted
evolution, or the coalescent process. Alternatively, it could reflect population
bottlenecks that occurred around the Plio-plestocene mass extinction, when up to 75% of
Caribbean species went extinct (around 3 million years ago) (Budd et al. 1994).
Although the available evidence appears to support the larval transport hypothesis,
the vicariance hypothesis cannot be ruled out. Each hypothesis has important
implications regarding the study of multigene families. If the origin of S. glynni was
from transportation across the Isthmus, then a rapid change in the proportion of ITS
haplotypes occurred. Genetic drift is the most likely explanation; however, inbreeding
and homogenization by concerted evolution may have also played a role. On the other
hand, if S. glynni originated by an ancient vicariant event, then the ITS region mutation
rate must be quite low, coalescent times are large and incomplete lineage sorting of
ancestral alleles has occurred.
This study is an illustration of some of the many problems that are associated with
identifying trans-isthmus geminate species pairs (reviewed by Marko 2002), and with
studying the boundary between population and species level processes. Methods such
as the nested clade analysis that are able to distinguish between historical events and
population level processes are necessary and valuable tools for examining these
processes, because it is exactly at this interface that the processes of speciation occurs
(Templeton 1998, 2001). Further empirical studies are necessary to examine how
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multiple-copy gene families behave within a population, and between recently diverged
species, in order to solve the S. glynni paradox.
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Table IV-1
Length variation, percent G + C content, number of individuals, number of
sequences, geographic region, collector and date for the ITS-1 and ITS-2 sequences
collected for this study. Abbreviations are as follows: EP, Eastern Pacific; ATL, Atlantic.
Collectors and dates are represented by numbers in superscript: 1 = J. Mate & H. Guzman
(01), 2 = E. Neves (00). The 5.8S gene had few polymorphisms, and a nearly constant
length of 106-107nt, and a 51% G+C content.
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ITS-1 ITS-2 Length Length No of No of Species Region (bp) %(G+C) SE (bp) %(G+C) SE Individuals Sequences S. siderea Panamá (ATL)1 305 44.22 0.35 192-193 53.41 0.58 3 13 S. radians Panamá (ATL)1 307 43.65 0.12 192 55.70 0.24 2 6 S. stellata Brazil (ATL)2 307-308 44.43 0.12 192 55.60 0.35 1 3 S. glynni Panamá (EP)1 304-305 44.67 0.12 192 53.09 0.27 4 16 Total 10 38
Table IV-1
138
Table IV-2
Matrix of averaged genetic distance between all molecular clones (in substitutions
per site, calculated by the Kimura 1980 method). Distances were calculated including
the entire ITS region (ITS-1, 5.8S, and ITS-2) implemented in MEGA 2.1 (Kumar et al.
2001).
139
g1c g1d
g1a, g2a g2d g3c g4d g4c s1a s1b s1d s2b s2d A B s3a r2b R ta tb tc
A haplotype network calculated by statistical parsimony, and the nested clade
design used for nested clade analysis. The network was estimated by the statistical
parsimony method implemented in TCS v 1.13 (Clement et al. 2000). The network
represents the set of 95% probable haplotype connections. Each rectangular small
circular node indicates a theoretical intermediate haplotype, the lines between indicate
one mutational distance. The nesting algorithm and rules outlined in Templeton et al.
(1987), and Templeton and Sing (1993). The procedure joins haplotypes separated by
one mutational event into 1-step clades proceeding from the exterior to the interior of the
network, all 1-step clades separated by one mutational event are joined into 2-step clades,
and so on…
149
Figure IV-3
3-2 3-1
g3c
g1c
g4c
s1b
s2d
s2c g2d g4d
s2b
s3a
s1a, s1e, s1f s2e, s2f
1-7
1-6 1-5 1-4
1-1
1-21-3
2-12-2
2-3
g1d
g1a, g1b, s1c g2a, g2b, g2c, s2a
g3a, g3b, s2c g4a, g4b, s3b
150
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V. Dissertation Conclusions
The ITS region is a promising molecular marker for a wide variety of studies in
Scleractinian coral. The majority of the phylogenetic and population genetic literature
is devoted to mitochondrial DNA, or highly conserved ribosomal genes; therefore, many
of the properties of ribosomal spacers are not well studied. The number of publications
on the subject are rapidly increasing, and the molecular marker has the potential to
revolutionize the fundamental understanding of what species are, how they are related to
each other, and how they change through time. The large technical problems, such as
problems with multiple sequence alignment, or intragenomic variability (due to
pseudogenes, separate chromosomal lineages, or incomplete lineage sorting) have the
potential to confound a study; however, these problems do not arise in many species, and
there are creative ways to overcome them.
A summary of the major conclusions of this dissertation follows: (1). In all species surveyed, intragenomic variation was low (less than 2% in P. lobata,
P. lobata-panama P. astreoides, P. colonensis, P. sverdrupi, P. panamensis, P.
divaricata, P. rus, P. furcata, S. stellata, S. radians, S. siderea, and S. glynni.).
Nucleotide diversity increased as individuals from distant regions were sampled, and
differences between species were at least an order of magnitude larger (12% or higher) in
all but a few closely related species, or species with questionable status.
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(2). According to alignment permutation: alignment ambiguities do not override the
underlying phylogenetic signal in comparisons between species, genera, and even
families, and the resulting phylogenies are consistent with previous molecular and fossil
studies.
(3). A putative cryptic species of P. lobata named P. lobata-panama was discovered
that is genetically and morphometricaly distinct from individuals collected across the
Pacific ocean from the Galápagos, Easter Island, Tahiti, Rarotonga, Fiji, and Australia.
(4). Patterns of gradual genetic and morphological differences between geographic
regions of P. lobata are consistent with isolation by distance, more specifically isolation
by ocean surface currents.
(5). ITS region mutation rates estimates from a wide variety of previously published
studies are surprisingly consistent, varying only approximately 5 fold. Small short-lived
organisms have much faster rates than large long lived ones.
(6). Due to sequences shared with S. siderea in the Caribbean, S. glynni could not have
originated from the Indo-Pacific. The available evidence, and a nested clade analysis,
suggest that S. glynni may have originated from a contemporary transport across the
Panamá canal. The alternative hypothesis of an ancient vicarient event cannot be