Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1998.Includes bibliographical references.by Ewann Agenbroad Berntson.Ph.D

Berntson, Ewann Agenbroad, 1968-

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Evolutionary patterns within the Anthozoa (phylum Cindaria) reflected in ribosomal gene sequences

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution April 1998This thesis project assesses phylogenetic relationships within the phylum Cnidaria,
at the subclass level within the Class Anthozoa, and at the ordinal level within the Subclass
Octocorallia. Traditional cladistics using morphological data have resulted in disagreements
over taxonomic relationships, primarily due to a paucity of morphological characters within
the Anthozoa and ambiguity about the significance of any given character. I have used
DNA sequence information to help resolve some of these issues. These phylogenetic
studies contribute to the understanding of divergence within the class Anthozoa.
Museum collections of preserved flora and fauna historically used for
morphological studies are now increasingly being utilized for addressing genetic questions.
The extraction of DNA from ethanol-preserved specimens of recent origin is practiced
routinely, but genetic analyses of long-preserved specimens have inherent difficulties due
to the slow degradation of DNA. The goal of this study was to demonstrate the feasibility
of isolating genomic DNA from museum specimens of octocorals and amplifying the
nuclear 18S ribosomal RNA gene. The DNA sequence for the complete 18S rRNA gene
can then be determined. Techniques were designed to solve several problems for obtaining
genetic sequences from museum specimens. The DNA extractions of museum specimens
yielded only small amounts of DNA of very low molecular weight, which limits the length
of Polymerase Chain Reaction (PCR) products that can be generated with standard
protocols. I was successful in producing PCR fragments from museum specimens by
performing an extended tissue digestion on the archival specimens, running an initial PCR
reaction, and then following with a reamplification of the original PCR product. The use of
taxon-specific PCR primers decreased the risk of amplifying contaminant DNA rather than
the target DNA in archival specimens. The combination of our modified extraction protocol
and PCR reamplifications with taxon-specific PCR primers allowed me to generate 700- to
1800-basepair sequences from 16 specimens from three different museum collections that
had been preserved for up to fifty years.
Taxonomic relationships within the corals and anemones (Phylum Cnidaria: Class
Anthozoa) are based upon few morphological characters: colony morphology and the
structure of the tentacles, gastric mesenteries, nematocysts, and skeletal axis. The
significance of any given character is debatable, and there is little fossil record available for
deriving evolutionary relationships. In this study I use complete sequences of 18S
ribosomal DNA to examine subclass-level and ordinal-level organization within the
Anthozoa. I investigate whether the traditional two-subclass system (Octocorallia,
Hexacorallia) or the current three-subclass system (Octocorallia, Hexacorallia,
Ceriantipatharia) is better supported by sequence information. I also examine the
phylogenetic affinities of the anemone-like species Dactylanthus antarcticus and the
putative antipatharian Dendrobrachia paucispina. Thirty-eight species were chosen to maximize the representation of morphological diversity within the Anthozoa. Maximum
likelihood techniques were employed in the analyses of these data, using relevant models of
evolution for the 18S rRNA gene. I conclude that placing the orders Antipatharia and
Ceriantharia into the Subclass Ceriantipatharia does not reflect the evolutionary history of
these orders. The Order Antipatharia is closely related to the Order Zoanthidea within the
Hexacorallia and the Order Ceriantharia appears to branch early within the Anthozoa, but
the affinities of the Ceriantharia cannot be reliably established from these data. My data
generally support the two-subclass system, although the Ceriantharia may constitute a third
subclass on their own. The Order Corallimorpharia is likely polyphyletic, and its species
are closely related to the Order Scleractinia. Dactylanthus, also within the Hexacorallia, is
allied with the anemones in the Order Actiniaria, and their current ordinal-level designation
does not appear to be justified. The genus Dendrobrachia, originally classified within the
Order Antipatharia, is closer phylogenetically to the Subclass Octocorallia. The 18S rRNA
gene may be insufficient for establishing concrete phylogenetic hypotheses concerning the
specific relationships of the Corallimorpharia and the Ceriantharia, and the branching
sequence for the orders within the Hexacorallia. The 18S rRNA gene has sufficient
phylogenetic signal, however, to distinguish among the major groupings within the Class
Anthozoa, and I can use this information to suggest relationships for several enigmatic
taxa. The Subclass Octocorallia (Phylum Cnidaria: Class Anthozoa) is comprised of the
soft corals, gorgonian corals, and sea pens. The octocorals have relatively simple
morphologies, and therefore few characters upon which to base taxonomic systems.
Historically, the Subclass Octocorallia was divided into seven orders: Helioporacea
(Coenothecalia), Protoalcyonaria, Stolonifera, Telestacea, Alcyonacea, Gorgonacea, and
Pennatalacea. It has been aigued that this an- angement exaggei ates the amount of
variability present among the species of the Octocorallia. The current taxonomy recognizes
the two orders of Helioporacea (blue corals) and Pennatulacea (sea pens), and assembles
the remaining species into a third order, Alcyonacea. The species within the Alcyonacea
exhibit a gradual continuum of morphological forms, making it difficult to establish
concrete divisions among them. The subordinal divisions within the Alcyonacea
correspond loosely to the traditional ordinal divisions. In this study I address the validity
of the historical ordinal divisions and the current subordinal divisions within the Subclass
Octocorallia. I also explore the phylogenetic affinities of the species Dendrobrachia
paucispina, which was originally classified in the Order Antipatharia (Subclass
Ceriantipatharia). Polyp stnicture indicates a closer affinity between Dendrobrachia and the
Subclass Octocorallia. I have determined the nuclear 18S rRNA sequences for 41 species
of octocorals, and use these to construct a molecular phylogeny of the subclass. I utilize
Maximum Likelihood techniques, employing a realistic model of evolution given these
species and this data set. The most likely trees from these sequence data do not support the
morphological taxonomy of the Octocorallia. The Order Pennatulacea is the most cohesive
group within the subclass, but is not monophyletic. The most likely trees indicate three
primary clades, one of which is undifferentiated and contains half of the species in this
analysis. These data cannot distinguish among the branching order of these three clades.
The morphological character of dimorphism (the presence of both autozooids and
siphonozooids within a single colony) corresponds loosely with the topology of the most
likely trees, and the monophyly of dimorphism cannot be rejected from these data. The
species Dendrobrachia paucispina has a close affinity with the genera Corallium and
Paragorgia (Alcyonacea: Scleraxonia), although its morphology suggests it is more similar
to the genus Chrysogorgia. The genetic divergence found within genera is approximately
equivalent to that found in other invertebrates, but the divergence found within families is
greater in the octocorals than in other invertebrates. This difference may reflect the
inappropriate inclusion of evolutionarily divergent genera within octocorallian families.
This study is more thorough than other anthozoan molecular phylogenetic studies to date. I have employed appropriate evolutionary models for maximum likelihood analyses, utilizing
complete 18S rDNA sequences from the majority of families within the Octocorallia. Many
of the relationships within the Octocorallia, however, remain ambiguous.The majority of my support came from the Office of Naval Research AASERT
grants #N00014-89-J-1431 and #N00014-95-1-0763. Thanks also to Peter Wiebe, who
let me piggy-back on his grant for the last AASERT. My next largest source of funding
was a NOAA/NURP fellowship through the Hawaiian Undersea Research Laboratory. I
also received support from the WHOI Education office, and a fellowship through the
Bermuda Biological Station

Evolutionary patterns within the Anthozoa (phylum Cindaria) reflected in ribosomal gene sequences

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