Phylogeny and Evolution of Anthopleura (Cnidaria: Anthozoa: Actiniaria)

Home , Actinia fragacea, Aeolidia papillosa, Aliciidae, Anthopleura, Anthopleura ballii, Aulactinia

Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Esprit Noel Heestand, B.A.

Evolution, Ecology, and Organismal Biology Graduate Program

The Ohio State University

2009

Thesis Committee

Dr. Marymegan Daly, Advisor

Dr. John Freudenstein

Dr. Andrea Wolfe

Esprit N. Heestand

2009

Abstract

Members of Anthopleura (Cnidaria: Anthozoa: Actiniaria) are some of the most well known and studied sea anemones in the world. Two distinguishing characteristics define the genus, acrorhagi and verrucae. Acrorhagi are nematocyst dense projections found in the fosse that are used for defense. Verrucae are suction cup-like protrusions on the column that hold rocks and small pebbles close to the anemone and prevent desiccation and DNA degradation. Previous studies have found that Anthopleura is non- monophyletic regards to Bunodosoma, another genus in Actiniidae. This study used molecular markers (12S, 16S, COIII, 28S) to circumscribe the polyphyly of Anthopleura, compared the informativeness of the four markers, and looked for patterns of evolution of acrorhagi and verrucae. This study shows that Anthopleura is polyphyletic regards to other genera within and outside of Actiniidae. It also shows that acrorhagi and verrucae are not valid characters when used to describe a monophyletic group, and did not find patterns of evolution of these two characters. The nuclear ribosomal marker 28S was the most informative marker and COIII was the least informative marker, however none of the markers had more then about 50% informativeness.

Acknowledgement

I would like to thank Abby Reft, Annie Lindgren, Derek Boogaard, Kody Kuehnl,

Jacob Olson, Joel McAllister, Luciana Gusmao, Reagan Walker, and Sarah Barath for all their help, counsel, encouragement, and reminding me that other things in the world exist besides this project, also, many thanks to my committee members for being so flexible and easy to work with. I would especially like to thank Dr. Meg Daly for taking a chance on me and accepting me as a Masters student; and Dr. Andi Wolfe for all her guidance, last minute help, and being a friend and teacher in all situations. I also thank my family for supporting me, believing in me, and commiserating with me thoughout this process.

Collection for this project was funded by the Fellowship for Graduate Student Travel from the Society for Integrative and Comparative Biology. The Cnidarian Tree of Life grant provided the funding for my lab work.

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Vita

2006………………………………………………B.A., The Ohio State University

2007………………………………………………Embryology, University of Washington

2008……………………………………………....Field Collection, La Paz Mexico

2008……………………………………….....…...Field Collection, Galveston, Texas

2007 – 2009……………………………………....M.Sc., The Ohio State University

Field of Study

Major Field: Evolution, Ecology and Orgamismal Biology Marine invertebrates: Anthozoans

Table of Content

Abstract………………………………………………………………………….…..…i

Acknowledgements……………………………………………………………..…...... ii

Vita…………………………………………………………………………..………....iii

Table of Content………………………………………………………..…………...….iv

List of Tables………………………………………………………………...………....vi

List of Figure…………………………………………………………………………....viii

Introduction…………………………………………………………………………..…1

Material and Methods………………………………………………….……………..…5

Study specimens…………………………………………………….…………..5

Molecular Methods………………………………………………………….….6

Data Analysis……………………………………………………………….…..7

Results…………………………………………………………………………………..10

Markers and congruence………………………………………………………...10

Maximum parsimony analysis…………………………………………………..10

Maximum likelihood analysis…………………………………………………...11

Constraint analysis……………………………………………………………….12

Comparative analysis………………………………………………………….…13

Discussion………………………………………………………………………..……15

Monophyly of Anthopleura…………………………………………….…..….15

Geographical clustering………………………………………….…….…...... 18

Sibling species………………………………………………………………….19

New species……………………………………………………………..…..….22

Geller and Walton 2001 analysis……………………………………………….23

Conclusion………………………………………………………………………....…...27

References…………………………………………………………………..……..……29

Appendix: Tables and Figures……………………………………………………….….34

List of Tables

Table 1. Taxa included in this analysis………………………………………………….35

Table 2. Markers used in this analysis…………………………………………………..36

Table 3. Attributes of markers…………………………………………………………..36

Table 4. Forced monophyly……………………………………………………………..36

Table 5. Comparative similarity of adjacent and sister taxa………………..……..…….37

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List of Figures

Figure 1. Defining characters of Anthopleura………………………………..………..38

Figure 2. Taxa and character list………………………………………………….…….39

Figure 3. Strict consensus tree from parsimony analysis…………………………...….40

Figure 4. Best maximum likelihood tree…………………………………………....….41

Figure 5. Geller and Walton analysis compared to current analysis……………...... …42

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Introduction

Anthopleura (Cnidaria: Anthozoa: Actiniaria) is one of the most familiar and well known genera of sea anemones. Members of Anthopleura are found on nearly every coast in the world and are the focus of behavioral and ecological research (e.g. Pineda and Escofet, 1984; Edmunds et. al., 1976; Francis, 1973; Francis, 1976; Bigger, 1980) because its species are locally abundant and tend to be locally diverse (Daly, 2004).

Species of Anthopleura reside in the intertidal zone, the area between the low tide and high tide water marks, which experiences one or two tide cycles a day, depending on the time of year and local conditions. The challenges these organisms face in this dynamic environment include desiccation, lack of oxygen, increased salinity, and extreme temperatures (e.g. Helmuth and Hofman, 2001). Rocky intertidal zones tend to be more species-rich, in terms of number of different species, than sandy intertidal zones

(Archambault and Bourget, 1996) due to available space (e.g. Dayton, 1971) for attachment and settlement.

Currently there are 46 valid species of Anthopleura (Fautin, 2007). Species of

Anthopleura vary from bright green in Anthopleura xanthogrammica to red in

Anthopleura balli to gray-brown in Anthopleura thallia. Size is also highly variable: adults of Anthopleura xanthogrammica, the giant green sea anemone, can have a diameter greater than 65 cm (Sebens, 1981). In contrast, Francis (1979) found that

1 members of Anthopleura elegantissima, the clonal sea anemone, tend to be smaller than solitary anemones. Members of Anthopleura have a varying number of tentacles; however, their tentacles are simple and hexamerously or irregularly arranged. Many members of Anthopleura have a symbiotic relationship with zooxanthellae and zoochorellae. The anemones provide the symbionce a substrate in which to safely live; in return the anemone is provided with fixed carbon that directly benefits the anemone (e.g.

Weis and Reynolds, 1998).

Duchassaing de Fonbressin and Michelotti (1860) described Anthopleura based on the distinct “pores” or verrucae (Figure 1) that cover the entire column of the anemone. Anthopleura krebsi, the type species of Anthopleura, also has a nematocyst- dense swelling in the margin of the column now known as an acrorhagus (e.g. Daly,

2003). These two characters are taxonomically important for genera within Actiniidae

(Carlgren, 1949). Anthopleura has both verrucae and acrorhagi. However Anthopleura is not monophyletic (McCommas, 1991; Geller and Walton, 2001; Daly, 2004; Daly et. al., 2008), implying that these characters are homoplastic.

In studies of comparative biology it is helpful to have monophyletic groups in order to examine patterns and processes of evolution. Anthopleura is an important experimental organism for studies of physiology (e.g. Jennison, 1978; Wicksten, 1984;

Pineda and Escofet, 1984), behavior (e.g. Edmunds et. al., 1976; Harris and Howe, 1979;

Francis, 1973; Francis, 1976), ecology (e.g. Lubbock, 1980; Bigger, 1980; Ayre and

Grosberg, 2005), and reproduction (e.g. Ford, 1964; Geller et. al., 2005). Anthopleura is also used for studying the symbiotic relationship between the photosynthetic

2 zooxanthellae and zoochlorellae and the anemone (e.g. Pearse, 1974; Saunders and

Muller-Parker, 1997; Verde and McCloskey, 2002; Weis et al., 2002).

Previous studies that found Anthopleura to be non-monophyletic (McCommas,

1991; Geller and Walton, 2001; Daly, 2004; Daly et. al., 2008) had limited taxonomic sampling. McCommas (1991) used 12 species from six genera to explore the relationships between genera in Actiniidae. He found one species of Anthopleura nested within a clade of Bunodosoma species. Geller and Walton (2001) sampled 13 species of

Anthopleura and one of Bunodosoma, and consistently found Bunodosoma nested within

Anthopleura. Daly (2004) found three species of Bunodosoma nested among 18 species of Anthopleura. With these smaller studies it is hard to understand the breadth of the polyphyly of Anthopleura and the circumscription of the group using the two diagnostic characters, acrorhagi and verrucae, that define Anthopleura. The previous studies (Geller and Walton, 2001, Daly, 2004) used, at most, three non-Anthopleura taxa in their analyses or limited sampling from within Anthopleura (McCommas, 1991; Daly et. al.,

2008).

In this study, DNA sequences were used to resolve the phylogeny of the genus

Anthopleura with respect to Aulactinia, Bunodosoma, Bunodactis, and Oulactis. These genera were sampled because they have acrorhagi, verrucae, pseudoacrorhagi, vesicles or a combination of these characters (Figure 2). Pseudoacrorhagi are swellings in the margin that do not have holotrichous nematocysts (Daly, 2003) and vesicles are non- adhesive rounded bumps or warts on the column (Daly, 2004). Descriptions of the genus were searched for the presence or absence of these characters (Figure 1). By sampling

Actiniidae with verrucae and acrorhagi I will better understand the circumscription and polyphyly of Anthopleura and whether these characters are useful for defining genera. I used molecular markers from both the nuclear (28S) and mitochondrial (12S, 16S, COIII) genomes to build an evolutionary tree and then mapped onto it the occurrence of acrorhagi, pseudoacrorhagi, verrucae, and vessicles.

Material and Methods

Study Specimens

Fifty-eight specimens representing eight families, 24 genera and 55 species were included in this analysis (Table 1). Specimens were collected by hand from the rocky intertidal zone and by SCUBA diving. Tissue for DNA analysis was taken from the tentacles or the pedal disk and preserved in 100% ethanol; the rest of the anemone was preserved in formalin. The specimens were identified by morphological characteristics and geographic location. Formalin preserved vouchers have been submitted to the

American Museum of Natural History (AMNH), the collection of Biodiversidad y

Ecología de Invertebrados Marinos (BEIM) at the University of Seville, the California

Academy of Science (CAS), the University of Kansas Natural History Museum

(KUNHM) and Raffles Museum of Natural History (RMNH). Sequences from previously studied specimens (Daly et. al., 2008) were downloaded from GenBank.

Only taxa for which three of the four markers were amplified have been included in the analyses. The outgroup consisted of Nematostellla vectensis and Hormathia armata. In addition to new sequences generated for this study, GenBank sequences for taxa with vouchers were used (e.g. Daly et. al., 2008). Voucher specimens were checked for identification if the placement of a taxon was questionable (Table 1). Sequences deposited in GenBank without a voucher (e.g. Geller and Walton, 2001) were used to

5 compare newly generated sequences to test, the informativeness of mitochondrial markers, and compare a small data sets to large data sets.

Molecular Methods

The Qiagen DNAeasy® kit (Qiagen Inc, Valencia, CA) was used to extract genomic DNA from ethanol-preserved tissue. Target sequences (12S, 16S, COIII, 28S) were then amplified using polymerase chain reaction (PCR) and published primers (Table

2) using Fisher-brand Taq. Each 25 µl reaction consisted of 1µl of template DNA, 0.5µl dNTP, 1µl Taq, 1µl forward primer, 1µl reverse primer, 1µl DMSO, 2.5 µl buffer, and

17µl water. The thermocycling profile was: (95 ºC for two min) + 30 × [(95 ºC for 15 sec) + (42 ºC for one min) + (72 ºC for one min)] + (72 ºC for three minutes). For genes that were not easily amplified, I used Herculase® (Stratagene, La Jolla, CA), following the manufacturer’s protocol. Each 25µl reaction consisted of 4µl of template DNA,

0.25µl Herculase, 0.5µl dNTP, 1.5µl forward primer, 1.5µl reverse primer, 2.5µl buffer, and 14.75µl water. The thermocycling profile was: (95 ºC for two min) + 10 × [(95 ºC for 30 sec) +(40 ºC for 30 sec) + (72 ºC for one min)] + 20 × [(95 ºC for 30 sec) + (42 ºC for 30 sec) + (72 ºC for one min)] + (72 ºC for seven min). Amplicons were cleaned with AmPure® (AgenCourt, Beverly, MA) magnetic bead solution and rehydrated to a concentration of 10ng/µl with deionized double-distilled water for sequencing. Cogenics in Houston, Texas, sequenced the clean amplicons. Sequencher 4.9 (Gene Codes

Corporation, Ann Arbor, MI) was used to trim, edit, and assemble forward and reverse

6 sequences. A BLAST search against GenBank was used to ensure the target sequence was amplified and not a symbiont or contaminant.

Data Analysis

Unaligned sequences for each marker were compiled in BioEdit Sequence

Alignment Editor (Hall, 1999) before being aligned in Muscle 3.6 (Edgar, 2007) using the default parameters (Table 2). The alignments were imported into Winclada (Nixon,

1999-2002) where leading and trailing gaps where trimmed, percent informative characters where found (Table 3), and the markers were combined into one matrix for further analysis. An Incongruence Length Difference (ILD) test (Farris et. al., 1994) was performed in Winclada to determine the combinability of the markers.

A parsimony analysis was run in TNT (Goloboff et. al., 2008) for each marker separately, for the mitochondrial markers, nuclear markers, and for all four combined markers. All analyses implemented the same parameters: gaps were missing data rather than a fifth state, and the minimum tree length was found ten times. Sectorial searches, the ratchet, and drift were implemented along with ten rounds of tree fusing to search the tree space. All analyses were subjected to 1000 rounds of jacknife resampling with 36% probability of removal; all nodes with less than 50% jackknife support were collapsed.

A parsimony search was performed under several constraints, including forced monophyly of taxa having verrucae, forced monophyly of taxa having acrorhagi, and forced monophyly of Anthopleura to determine if there were suboptimal trees with these characters as a monophyletic group. The maximum parsimony trees found in TNT were

7 used as null hypotheses for testing alternative hypotheses in terms of tree length and the number of extra steps it would take to reveal monophyletic groups with morphological synapomorphies. I did this by visually comparing trees produced by they constraint analysis to the maximum parsimony trees.

ModelTest 3.7 (Posada and Crandall, 1998) was run in PAUP (Ver. 4.03,

Swofford, 1999) to find the model used in the Randomized Axelerated Maximum

Likelihood (RAxML) (Stamatakis, 2006) analysis. The GTR + gamma model was chosen based on the Akaike information. The same model was chosen for both the mitochondrial and nuclear partitions, because there was no incongruence in the separate gene matrices based on the ILD test. The model was used in RAxML using CIPRES

Portal v 1.5 (Stamatakis et. al., 2008). The resulting data sets were then subjected to

1000 rounds of bootstrap resampling using sample with replacement. Phylip (Ver. 3.67,

Felsenstein, 2007) was then used to combine the resulting bootstrap trees.

In PAUP, a neighbor-joining search was implemented using the TRN + G model

(alpha = 0.4) to generate trees to compare with those from a previous study (Geller and

Walton, 2001) in order to ensure correct identification of the taxa and sequences from both studies. Sequences from Geller and Walton (2001) were downloaded from

GenBank and combined in a matrix with the new sequences generated in this study.

Sequences generated from this, analysis which had a conspecific counterpart in the Geller and Walton analysis, were also included in the matrix. A parsimony analysis and jackknife analysis were run on the matrix in TNT using the same criteria as above, to compare the relationship of species within Anthopleura over a large dataset and small

8 data set, and to examine the influence different gene loci have when determining the relationships among the taxa. The combined data set was also subjected to a maximum likelihood analysis using RAxML and a bootstrap analysis was performed to determine support. All trees were viewed in FigTree v1.2.2 (Rambaut, 2006-2009).

Winclada was used to measure the similarity between two sequences. The compared taxa were chosen based on their position in the tree and inferred relationships between sister taxa. For example, sequences of Gyractis sesere were compared to determine the percent similarity between the sequences derived from different DNA accessions. The distance between each marker was measured individually and then across the combined markers. The outgroup was compared to the basal most member of the ingroup as a baseline for the compared distances.

Results

Markers and congruence

The maximum length of the markers ranged from 657 to 998 bases. Their aligned length ranged from 604 to 1500 (Table 2). Partial 28S was the longest marker and the most variable (Table 2), and also required the most gaps to align. Cytochrome oxidase

III (COIII) was the shortest marker and required no gaps in the alignment; it was also the least informative of the markers in this analysis, having the lowest percentage of parsimony informative characters. No comparison was found to be significantly incongruent based on the ILD test (p ≤ 0.05). There was very little resolution in the trees produced by 16S, the strict consensus tree had only 22 resolved nodes (Table 3). In contrast the strict consensus tree from 28S had 54 resolved nodes.

The markers used in this analysis provide support from the basal to middle section of the tree, but not for the terminal branches. There is low support for the distal nodes because there were few differences at the species level. Mitochondrial markers are known to accumulate mutations more slowly then nuclear markers in anthozoans

(Shearer et. al., 2002).

Maximum parsimony analysis

Twelve equally parsimonious trees were found from the analysis of combined sequences. Individual trees agreed in overall topology in that they all showed a

10 polyphyletic Anthopleura (Figure 3), however the internal relationships between the taxa were variable. Species from Anthopleura were intermixed within other genera and families. Due to the conflict terminal nodes, the strict consensus tree shows little resolution for a majority of the tree. The strict consensus tree has six clades of three or more taxa. There are five clades of particular interest: a small clade with four taxa that is highly supported (box C); a clade of two species of Anemonia and one Isoaulactinia (box

B1); a well supported clade of species from Pseudactinia (box B2); a clade of non-

Anthopleura Actiniids (box D); and the largest clade, which has six genera and is highly internally supported (box E). This clade includes members from Anthopleura,

Anthostella, Aulactis, Bunodactis, Bunodosoma, and Oulactis, which are the focal genera for this study. Three species of Anthopleura are the basal ingroup taxa. Another group found on this tree is unresolved in their relationships (box A). This group is resolved in the maximum likelihood tree.

Maximum likelihood analysis

Many of the same relationships seen in the maximum parsimony tree were also seen in the maximum likelihood tree (Figure 4). Clade A shows a monophyletic group that was not resolved in the maximum parsimony analysis (Figure 3). Clades B1 and B2 are united (box B) in the maximum parsimony analysis (Figure 3). The maximum likelihood tree also has clades C, D, and E resolved as a monophyletic group (Figure 4).

Clade A (box A) in the maximum likelihood tree includes members of nine genera, three families, and members of Anthopleura (Figure 4). In contrast, clade B (box B) does not

11 contain any members of Anthopleura, but includes members from three separate genera:

Anemonia, Isoaulactinia, and Pseudactinia. The same three species of Anthopleura are found at the base of the ingroup (Anthopleura pacifica, Anthopleura midori, and

Anthopleura xanthogrammica).

The branches for the members of Pseudactinia are long, however the branch that unites these species is also long. This clade is not an example of long branch attraction as these species form a monophyletic group in the maximum parsimony analyses as well.

When members of this clade are excluded from the analysis the remaining taxa still cluster together. Similarly clade C (box C) in the maximum likelihood analysis does not show long branch attraction. When members of this clade are excluded from the analysis, the other taxa still form a monophyletic group instead of moving in the tree.

The maximum likelihood tree is more resolved than the maximum parsimony tree, and has two more nodes with support over 50%. The maximum parsimony tree has a higher percentage of nodes with high support (over 90%) compared to the maximum likelihood tree. There are variable branch lengths thoughout the maximum likelihood tree. The clustering of clade D is not due to long branch attraction since Aulactinia stella is the only member of that clade with a long branch.

Constraint analysis

A constraint analysis was performed to determine the validity of Anthopleura being non-monophyletic by determining how much longer a tree would be when forcing taxa with acrorhagi and/or verrucae into a monophyletic group. In the analysis, there are

36 taxa that have verrucae and 39 taxa that have acrorhagi, about half the taxa in the analysis have both these characters. The analysis produced about 10 times more parsimonious trees than the maximum parsimony analysis. Each tree from the constraint analysis was almost twice as long as the most parsimonious trees found (Table 4).

Comparative analysis

A matrix consisting of 15 sequences from Geller and Walton (2001) and nine sequences generated for this study were used in a maximum parsimony and maximum likelihood analyses to evaluate the results from this study. A parsimony analysis of this matrix found 25 equally parsimonious trees with 26 steps. Four of the taxa from this analysis came out as ‘sisters’ to the corresponding species from the Geller and Walton

(2001) analysis. Three of the taxa from this analysis came out adjacent to the corresponding species from the Geller and Walton (2001) analysis. Two of the taxa from this analysis did not match their Geller and Walton (2001) counterpart (Figure 5A). The overall topology of the tree is similar to that found by Geller and Walton (2001), except for the placement of the taxa that did not group with their counterparts: Anthopleura elegantissima and Anthopleura kurogane.

Maximum likelihood analysis of this matrix recovered a slightly different topology than the maximum parsimony strict consensus tree (Figure 5B). Six of the nine taxa from the current analysis pair with the similarly named taxa from the Geller and

Walton (2001) analysis either adjacent or as sister taxa. In addition to the two taxa

(Anthopleura elegantissima and Anthopleura kurogane) that do not pair with their

13 conspecific counterpart in the parsimony analysis, Anthopleura dowii fails to match its counterpart. Anthopleura dowii is resolved in the clade containing Bunodosoma cavernata. In contrast, both sequences of Anthopleura dowii are collapsed along the basal branch and adjacent to each other in the maximum parsimony tree. The branch lengths throughout the tree are distributed evenly among the taxa.

Nine sequences from the current study that match the taxon sampling from Geller and Walton (2001) were used in a neighbor joining analysis to compare results from the two studies. The topology of the neighbor joining tree (not shown) matches that of Geller and Walton (2001). Neither of the neighbor joining trees matches the topology of the maximum parsimony and maximum likelihood trees. This may be the result of differences in the tree building methodologies or an artifact of limited taxon sampling.

Discussion

Monophyly of Anthopleura

Previous studies (McCommas, 1991; Geller and Walton, 2001; Daly, 2004; Daly et. al., 2008) have shown that several families and genera are nested within the genus

Anthopleura. For example, members of Bunodosoma, Bunodactis, and Aulactinia are nested within Anthopleura and they do not have both acrorhagi and verrucae; instead they have a variation of these structures. These previous studies aimed to understand the relationships between species within Anthopleura (Geller and Walton, 2001; Daly, 2004) or reconstruct the overall evolutionary framework of Actiniaria (McCommas, 1991; Daly et. al., 2008). The maximum parsimony and maximum likelihood analysis agree that

Anthopleura is polyphyletic.

Although Geller and Walton’s (2001) analysis was not as extensive as the current analysis, they also found a non-monophyletic Anthopleura. Geller and Walton (2001) sampled 13 species of Anthopleura and one of Bunodosoma, and consistently found

Bunodosoma within Anthopleura. Daly’s (2004) tree is in disagreement with Geller and

Walton’s (2001) tree with regard to the relationships between the species of Anthopleura, but she too found species of Bunodosoma nested among 18 species of Anthopleura.

McCommas (1991) used 12 species from six genera to explore the relationships between different genera in Actiniidae. He found one species of Anthopleura nested within a clade of Bunodosoma, which is also similar to the findings of Geller and Walton (2001).

The disagreements between the trees from these studies may be due to different taxonomic sampling and sample size. However, even though each of these phylogenies has a relatively small sample, each found Anthopleura to be non-monophyletic.

Daly et. al. (2008), in a broad study of Actiniarian phylogeny, found species of

Anthopleura scattered across a monophyletic endomyarian clade intermixed with

Bunodactis, Actinia, and Anemonia. In this tree, a close relationship between

Anthopleura and species from families outside of Actiniidae is seen. Within the endomyarian clade there are many species scattered within it that have verrucae, vesicles, pseudoacrorhagi, and acrorhagi. In the current analysis these characteristics also do not seem to characterize distinct clades.

The characters that define Anthopleura, the presence of acrorhagi and verrucae, are homoplastic. The tree indicates that acrorhagi and verrucae have been lost and re- evolved multiple times because taxa with these characters (Anthopleura pacifica,

Anthopleura midori, and Anthopleura xanthogrammica) are at the base of both the maximum parsimony and maximum likelihood trees. However, there is no recognizable pattern of transformation from acrorhagi to pseudoacrorhagi or verrucae to vesicles when these characters are mapped onto the maximum parsimony tree and maximum likelihood tree (Figure 3 and 4). Neither of these characters describes a monophyletic group individually or together; thus they are not reliable diagnostic characters for a genus.

When Anthopleura is forced to be monophyletic, the best trees are almost twice as long as the best tree with no constraints (Table 4). There is no phylogenetic support for

Anthopleura being a monophyletic group, for verrucae circumscribing a monophyletic group, or for acrorhagi circumscribing a monophyletic group.

Polyphyly for Anthopleura lends support to several hypotheses about the genus.

First, it suggests that the characters that describe Anthopleura, adhesive verrucae and acrorhagi, are adaptive and provide an advantage in the intertidal zone. Verrucae hold pebbles and shells to the column, trapping water and preventing desiccation due to wind and heat (Hart and Crowe, 1977). The debris adhering to the column may also act as a sunscreen, preventing DNA degradation and mutations (Dykens and Shick, 1984). Acrorhagi are primarily used for defense of the individual and of the clonal group (Abel, 1954; Bonnin,

1964). Having both or even just one of these characters causes the anemone to be more competitive in its dynamic intertidal environment where space is a limiting factor.

Secondly, the parsimony tree suggests that these characters have been lost several times though out the history of these organisms. Verrucae have been lost 22 times and acrorhagi have been lost 20 times. Since neither of these characters describe clades it seems that acrorhagi and verrucae are easily lost and gained.

This analysis does not look in depth at the morphology of these characters, but instead relies on the observations of others (e.g. Carlgren, 1940; Daly, 2003; Daly and den Hartog, 2004). For example, Oulactis and Anthopleura are both described as having acrorhagi and verrucae but members of Oulactis have small, tightly packed verrucae on small, frond-like lobes of the column (Carlgren, 1949), which is different from the verrucae Anthopleura is described as having. Imprecise descriptions of characters results in non-homologous characters classified as homologous ones.

Geographical clustering

Daly’s (2004) analysis looked into the historical factors underlying the distribution of Anthopleura and its allies. She sampled 21 ingroup taxa, 18 of which were species of Anthopleura, and constructed a morphological matrix of attributes. The resulting evolutionary tree was divided into two major clades: a Southern Hemisphere basal clade and a Northern Hemisphere clade. Members of the Southern Hemisphere clade were wide spread species and taxa that were only found in the Southern

Hemisphere. Members of the Northern Hemisphere clade were taxa that were only found in the Northern Hemisphere, including European species of Anthopleura. European species of Anthopleura did not cluster together.

When reconstructing Daly’s (2004) tree using sequences generated from this analysis that had corresponding taxa in her analysis I did not find the same evolutionary tree, nor the same major geographical clustering. Instead of finding clades that separate into Northern Hemisphere and Southern Hemisphere, several local groupings were found.

For example, some of the species from Japan group together (e.g. Stichodactyla gigantea and Heteractis magnifica), and I found a small clade of species from Oman (Anthopleura dixoniana and Heteractis aurora). Specimens from other areas, like the Pacific coast of

North America, did not cluster together but instead were scattered thoughout the tree (e.g.

Anthopleura elegantissima and Anthopleura xanthogrammica). I can also see this from specimens from Europe (e.g. Anthopleura biscayensis, and Anthopleura thallia). The clustering could be an artifact of sampling heavily in these areas. Many of the specimens used for this analysis were collected from Japan (12%), Oman (8%), and South Africa

(20%). Due to chance alone, there is greater likelihood that organisms from these locations will cluster together because they make up a large part of the sampled taxa.

Species of Bunodosoma and Aulactinia from South Africa (Bunodosoma capensis and Aulactinia reynaudi) come out in a clade with species of Anthopleura from South

Africa (Anthopleura mariscali, Anthopleura anneae, Anthopleura insignis, and

Anthopleura michaelseni). Bunodosoma has acrorhagi and no verrucae (Carlgren, 1949) and Aulactinia has verrucae and no acrorhagi (Carlgren, 1949). This suggests that the location of the anemone, geographically, is just as important when identifying it as other physical characteristics of the anemone for some localities like South Africa. Because

Anthopleura is generally species-rich when present, it is still important to have characters to distinguish one species from another. For example, when identifying organisms it is important to have a complete description (e.g. nematocyst types, fertility, and description of mesenteries and sphincter) however, the presence or absence of acrorhagi and verrucae are not useful for describing a monophyletic genus.

Sibling species

Species of Gyractis sesere were sampled from geographically distant locations

(Hawaii, South Africa, U.S. Virgin Islands) (Table 1). One specimen of Gyractis excavata from Australia was also included. Only two of the samples were included in the analysis because all samples were found to be genetically similar, with a total of 12 base pair differences across all four markers (12S, 16S, COIII, 28S) in the four specimens, an overall similarity of 99.6%. A maximum parsimony analysis was performed and all

19 species of Gyractis came out as a monophyletic group with 100% jackknife support (not shown). Based on the phylogenetic species concept, which was later added to by Cracraft

(1989) and called the diagnostic concept, I consider all four of these samples to belong to the same species. This molecular evidence supports the morphological evidence that

Gyractis sesere is the same species as Gyractis excavata (Fautin, 2005) because the specimen collected from Australia grouped with the other three specimens. This cosmopolitan species would be a good model for studing gene flow across the world’s major oceans.

Anthopleura dowii has two forms, solitary and clonal. The solitary form tends to be larger and live lower in the intertidal zone than the clonal Anthopleura dowii form

(personal observation). A similar phenomenon to this is seen on the Pacific Coast of

North America with Anthopleura elegantissima and Anthopleura sola. These two species were thought to be the same species, Anthopleura elegantissima, with two different forms, solitary and clonal. McFadden et. al. (1997) determined the forms to be two different species based on ten highly polymorphic allozyme loci and Pearse and Francis

(2000) declared them two different species, Anthopleura elegantissima and Anthopleura sola, based on the genetic and behavioral differences. The analysis of the four genes shows that these two forms of Anthopleura dowii are always siblings (Figure 3 and 4) with 11 base pair differences between the five anemones sequenced (three from separate clones and two solitaries), an overall similarity of 99.7%. These samples were taken from two locations approximately five kilometers apart, although Anthopleura dowii ranges from southern California to Panama (Fautin, 2007). Given the variation in

20 geographically close populations, as compared to the genetic similarity found in Gyractis sesere (99.6% globally), Anthopleura dowii has the potential to be very genetically diverse.

In every individual gene tree and in the combined analysis, Anthopleura krebsi and Anthopleura pallida are sister taxa, with Anthopleura waridi as their sister (Figure 3 and 4). It is unclear whether Anthopleura krebsi and Anthopleura pallida are two distinct species. Daly and den Hartog, (2004) were able to describe several morphological differences between the two species, but I found no base pair differences between the two taxa across four markers. Because these two species are identical for these markers, they are either the same taxon or there must be extensive gene flow between them. Based on the phylogenetic species concept and diagnostic concept they are the same species because they always form a monophyletic group. The biological species concept states that species are groups of natural interbreeding populations (Mayr, 1970); based on this concept these two taxa are considered the same species. Alternatively, one of the specimens could be misidentified resulting in the sequencing of identical species.

Daly and den Hartog (2004) found three clear differences between Anthopleura krebsi and Anthopleura pallida: column color, shape, and verrucae arrangement. As previously discussed, verrucae are homoplastic and not a reliable character for defining this group. Color in anemones can be a confusing character for describing species because a single species could have several very different color morphologies, as with

Phymactis clematis (Drayton in Dana, 1846). Because the characters that differentiate the two species are so variable, I conclude that the three distinct morphological

21 differences observed between these two species are different varieties of the same species, Anthopleura krebsi. However, this is based on sequencing two specimens, further genetic testing needs to be done to explore this.

Carlgren (1940) described two species of Anthopleura from South Africa,

Anthopleura anneae and Anthopleura insignis. He proposed that Anthopleura insignis

(preserved) was identical to Anthopleura michaelseni, a species he previously re- described from South Africa (Carlgren, 1938). Carlgren (1940) based the description of

Anthopleura insignis on the differences in the marginal sphincter, which is more diffuse in Anthopleura insignis. These species also differ in the size of their nematocysts in their filaments, which is how I identified the two taxa. I found 11 base pair differences out of

3353 total base pairs, which is 99.7% overall similarity (Table 5). In these analyses, they are sister species (Figure 3 and 4). In the individual gene trees and combined gene trees they come out as siblings but there is less than 50% jackknife support for their branch. I conclude that these are two distinct species based on the markers used in this analysis.

New species

Based on this analysis and inspection of the collected organisms there is one undescribed species in South Africa: “Pseudactinia sp. South Africa.” From a physical examination of the anemones in this study, it is impossible to assign it to one of the described species because it does not have the same number of tentacles and mesenteries as the described species (both having more than the described number), it is colored differently than the species described by Carlgren (1938), and has lower genetic

22 similarity compared to other sibling species (Table 5). The phylogenetic species concept and diagnostic concept states that same species form a monophyletic group and

Pseudactinia sp. South Africa is not sister to either of the known species, but is sister to the well supported sister group, which include other members of Pseudactinia from

South Africa.

The new species of Pseudactinia from South Africa is completely orange except for the purple tips of the tentacles. This color combination is not mentioned in the description of the other two Pseudactinia species from South Africa (Carlgren, 1938). In the maximum likelihood tree three species in Pseudactinia come out together with 100% bootstrap support. In the maximum parsimony tree they also form a single monophyletic group and have 99% support. There are 298 base pair differences between Pseudactinia flagellifera and Pseudactinia sp., which is 91.1% overall similarity between the two species. Between Pseudactinia varia and Pseudactinia sp. there are 295 base pair differences, which results in 91.2% overall similarity.

Geller and Walton 2001 analysis

Geller and Walton’s (2001) analysis used two genes, 16S and COIII, to build a phylogeny of 13 species of Anthopleura and one species of Bunodosoma to better understand the origins of clonal growth and symbiosis in this genus. They concluded that there have been a number of shifts between being clonal and solitary between species of

Anthopleura. There also seem to be gains and losses of symbiosis. Geller and Walton

(2001) hypothesized Anthopleura as being polyphyletic based on one non-Anthopleura taxon being resolved in the ingroup instead of clustering with the outgroup.

When comparing the sequences from the current analysis to Geller and Walton’s

(2001) analysis of Anthopleura, six of the nine samples consistently grouped together

(Figure 5). Some nodes were well supported (e.g. Bunodosoma cavernata), whereas others had less than 50% support (e.g. Anthopleura xanthogrammica). The varying support for these species could be due to the number of characters that unite the members. Bunodosoma cavernata have one more character uniting the two taxa than

Anthopleura xanthogrammica (Table 5).

Anthopleura dowii is not located beside its counterpart in the maximum likelihood analysis; in the maximum parsimony analysis the two taxa are adjacent to each other

(Figure 5). This could be due to the variability of Anthopleura dowii. Specimens from the current analysis were collected from locations that were close very together, resulting in sequences that were very similar. It is unlikely that Geller and Walton collected specimens from the same location as I did. As discussed above, this is a highly variable species. When comparing the sequences from the two analyses, I found 1028 out of 1052 aligned bases, base pair similarity between the two organisms, this amounts to 97.7% similarity (Table 5). Even though they are not resolve as ‘sisters’ I think they are still the same species and differences I see between these two sequences are population level differences.

Anthopleura elegantissima and Anthopleura kurogane are also two species where our samples do not correspond to their Geller and Walton (2001) counterparts (Figure 5).

When comparing the two most parsimonious trees (not shown), these two taxa jump around within the tree and do not group with their counterparts, hence when the strict consensus tree is produced their branch is collapsed to the most recent common ancestor.

When comparing the voucher with the description of the organism it seems that they are correctly identified.

Comparing the combined (16S and COIII) sequences of Anthopleura elegantissima to sequences downloaded from GenBank from Geller and Walton’s (2001) analysis there are 15 base pair differences between the two analyses, which amounts to

98.5% similarity (Table 5) between the two sequences. When doing the same comparison with the sequences generated for Anthopleura kurogane and Geller and

Walton’s (2001) analysis sequences one base pair difference between the two sequences was observed, which results in 99.9% similarity (Table 5). Both of the sequences generated for this analysis are very similar to the sequences used in Geller and Walton’s

(2001) analysis. There are two reasons why these taxa jump around in the tree and do not group with their conspecific counterpart. First, there could be some characters that are in conflict so when the strict consensus is generated the branches are collapsed. Second, there could be not enough support to unite the two taxa. When comparing the outgroup with a member of the ingroup there is 96.5% overall similarity (Table 5) between the two, which is 36 base pair differences.

The genes used in this analysis (16S and COIII) are informative at the root of the tree but not the distal branches of the tree (Figure 5). This contributes to the low diversity between the conspecific taxa and does not provide sufficient differentiation

25 between the species. In order to have high support for the distal branches of the tree a marker has to be used that is more specific between closely related species.

The analysis of Geller and Walton (2001) contains a small sample size and contains taxa that do not form a monophyletic group as seen in this study. When the nine taxa that are used in the comparison of the Geller and Walton (2001) analysis are used to build a maximum parsimony tree the same relationships are seen between the taxa as seen in Geller and Walton’s (2001) paper. I also found Anthopleura to be non-monophyletic but a larger sample size is needed to totally circumscribe the problem.

Conclusion

This study represents the most thorough analysis of Anthopleura ever conducted.

The major finding is that Anthopleura is polyphyletic as currently circumscribed.

Previous studies had sample sizes that were too small to fully characterize polyphyly of

Anthopleura. It is important to describe monophyletic groups to better understand the evolutionary relationships between these organisms and to be able to extrapolate experimental results using the correct sibling species. Unfortunately, the characters that describe Anthopleura do not yield a monophyletic group, which indicates the need for taxonomic revision of the genus.

Geographic patterns from previous analyses (Daly, 2004) were not supported in this analysis, but local geographic clustering was revealed (Figure 3 and 4). This could be due to recent speciation events in the area or sampling bias. Many of the specimens were collected from similar locations. Gyractis sesere is a cosmopolitan species where all taxa sampled are genetically very similar. This is likely due to a slow rate of genetic mutation or gene flow between populations of this globally dispersed species.

Anthopleura krebsi and Anthopleura pallida are genetically similar but morphologically different. Based on field identification characters they are separate species (Daly and den

Hartog, 2004), however, here they are a single species (Anthopleura krebsi) using the phylogenetic species and biological species concepts. Two species, Anthopleura michaelseni and Anthopleura insignis, are genetically different species. This is in

27 agreement with the Carlgren (1940) presumption that they are different species. A new species of Pseudactinia is found based on these analyses. It is genetically distinct from other species of Pseudactinia found in the location of collection.

I was only able to collect half the number of known valid species of Anthopleura.

In order to fully understand the nature of polyphyly in Anthopleura it would be necessary to include more taxa from both Anthopleura and from other genera in Actiniidae. It is necessary to sample broadly since acrorhagi and verrucae are characters that do not define monophyletic groups. Combining morphological analysis with a genetic analysis would ensure classification of morphological characters (e.g. verrucae and acrorhagi) as correct. By sampling broadly it would be possible to understand the evolution of these characters. New markers need to be developed for distinguishing between anemone species. The markers used in this analysis are too broad to ‘sort out’ species level relationships with high support.

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Appendix: Tables and Figures

Family Species Collection locations Voucher Actiniidae Actinia fragacea Northern France CAS Anemonia erythraea South Africa KUNHM Anemonia viridis Northern France CAS Anemonia natalensis South Africa KUNHM Anthopleura anneae South Africa KUNHM Anthopleura ballii Northern Ireland KUNHM Anthopleura biscayensis Southern England KUNHM Anthopleura buddemeieri Singapore RMNH Anthopleura dixoniana Oman KUNHM Anthopleura dowii (clonal) Baja California Sur, Mexico AMNH Anthopleura dowii (solitary) Baja California Sur, Mexico AMNH Anthopleura elegantissima California, USA AMNH Anthopleura inornata Honshu, Japan KUNHM Anthopleura insignis South Africa KUNHM Anthopleura japonica Honshu, Japan KUNHM Anthopleura krebsi Florida, USA KUNHM Anthopleura kurogane Korea KUNHM Anthopleura mariscali Ecuador KUNHM Anthopleura michaelseni South Africa KUNHM Anthopleura midori Honshu, Japan KUNHM Anthopleura pacifica Honshu, Japan KUNHM Anthopleura pallida Florida, USA KUNHM Anthopleura sp. Ecuador KUNHM Anthopleura thallia Engand KUNHM Anthopleura waridi Oman KUNHM Anthopleura xanthogrammica California, USA KUNHM Anthostella stephensoni South Africa KUNHM Aulactinia incubans Washington, USA AMNH Aulactinia marplatensis Argentina AMNH Aulactinia reynaudi South Africa KUNHM Aulactinia stella Maine, USA KUNHM Bunodactis verrucosa Northern Ireland KUNHM Bunodosoma capensis South Africa KUNHM Bunodosoma cavernata Texas, USA AMNH Bunodosoma grandis Ecuador KUNHM Bunodosoma granulifera US Virgin Islands AMNH Cnidopus japonicus Hokkaido, Japan KUNHM Epiactis lisbethae Washington, USA KUNHM Gyractis sesere South Africa KUNHM Gyractis sesere US Virgin Islands AMNH Isoaulactinia hespervolita Baja California Sur, Mexico AMNH Isosicyonis striata Antartica BEIM Macrodactyla doreenensis Australia KUNHM Oulactis muscosa Australia KUNHM Phymactis clematis Baja California Sur, Mexico AMNH Pseudactinia flagellifera South Africa KUNHM Pseudactinia sp. South Africa KUNHM Pseudactinia varia South Africa KUNHM Urticina coriacea California, USA KUNHM Actinodendridae Actinostephanus haeckeli Indonesia KUNHM Aliciidae Triactis producta Oman KUNHM Liponematidae Liponema brevicornis Washington, USA USNM Phymanthidiae Phymanthus loligo Oman KUNHM Stichodactlyidae Heteractis aurora Oman KUNHM Heteractis magnifica Honshu, Japan KUNHM Stichodactyla gigantea Honshu, Japan KUNHM Edwardsiidae Nematostella vectensis Maryland, USA AMNH Hormathiidae Hormathia armata Antartica BEIM

Table 1: Taxa included in this analysis. Species name, Location of collection of organism, and Voucher location.

Marker Primer source # of Max length Aligned sequences (bp) length (bp) 12S Chen et. al. (2002) 56 966 1142 16S Geller and Walton (2001) 58 935 1221 CoIII Geller and Walton (2001) 58 657 604 28S Chen and Yu (2000) 53 998 1500

Table 2: Markers used in this analysis. Primer source, the number of taxa in the analysis for each marker, the maximum length of sequenced site, the aligned length after alignment in Muscle, and the model chosen for the matrix in Modeltest.

Marker % informative # parsimonious # of nodes Number of characters trees collapsed in steps (tree consensus tree length) 12S 28.47% 23 30 682 16S 36.17% 27 37 372 CoIII 26.48% 91 11 384 28S 50.93% 4 29 3066 Combined 31.18% 12 27 4954

Table 3: Attributes of markers. Percent of informative characters in the aligned matrix based on the individual markers and the combined analysis, the number of equally parsimonious trees found in each analysis, the number of nodes collapsed when the strict consensus tree was built, and the number of steps in the most parsimonious trees.

Trait being constrained into Number of trees Number of steps a monophyletic group Anthopleura 290 8953 Verrucae 1122 8687 Acrorhagi 111 9005 Maximum parsimony trees 12 4954

Table 4: Forced monophyly. The results of the constraint analysis when forcing different taxa to form monophyletic groups based on the presence of acrorhagi or verrucae or both.

Species compared % Similarity Overall 12S 16S COIII 28S Focal Anthopleura krebsi and 100% 100% 100% 100% 100% organisms Anthopleura pallida compared Anthopleura dowii clonal 99.7% 100% 99.1% 100% 99.5% to each and solitary other Gyractis sesere global 99.6% 100% 100% 100% 98.9% Aulactinia marplatensis 99.6% 100% 100% 100% 97.6% and Anthopleura biscayensis Anthopleura michaelseni 99.6% 100% 100% 100% 99.0% and Anthopleura insignis Anemonia erythraea and 98.1% 100% 99.2% 99.0% 96.0% Anemonia natalensis Anthopleura ballii and 97.7% 100% 99.3% 100% 94.4% Anemonia viridis Pseudactinia sp. and 91.2% 100% 87.7% 98.9% 85.5% Pseudactinia varia Pseudactinia sp. and 91.1% 99.8% 87.6% 98.7% 88.6% Pseudactinia flagellifera Nematostella vectensis 90.9% 94.9% 95.9% 94.5% 80.7% and Anthopleura pacifica Geller and Anthopleura carneola 100% 100% 100% Walton and Anthopleura krebsi taxa Bunodosoma cavernata 100% 100% 100% compared Anthopleura 99.9% 100% 99.8% with taxa xanthogrammica from Anthopleura kurogane 97.8% 99.2% 97.4% current Anthopleura dowii 97.7% 99.6% 96.1% study Anthopleura 97.3% 100% 96.0% elegantissima Epiactis prolifera and 93.1% 95.3% 92.7% Anthopleura elegantissima

Table 5: Comparative similarity of adjacent and sister taxa. Percent similarity of focal taxa over the four gene regions (12S, 16S, COIII, 28S). Geller and Walton compared to current analysis. Percent similarity between conspecific taxa over both gene regions. Squares are left blank when there are no markers to compare.

Figure 1: Defining characters of Anthopleura. Photograph of Anthopleura elegantissima with line drawings of a suction cup-like verruca (Stephenson, 1928) and an acrorhagus (Stephenson, 1928).

Figure 2: Taxa and characters. Species included in the analyses and the characters that they have (acrorhagi, pseudoacrorhagi, verrucae, and vesicles).

Figure 3: Strict consensus tree from the three most parsimonious trees for all four genes (12S, 16S, COIII, 28S) from 58 taxa. Jackknife values above 50% are indicated. Species with verrucae marked (*), species with acrorhagi marked (+), species with vesicles marked (&), species with pseudoacrorhagi marked (^). 40

Figure 4: Best maximum likelihood tree. This is found from the maximum likelihood analysis for all four genes (12S, 16S, COIII, 28S) from 58 taxa. Bootstrap values are written on the tree when a clade is supported by more than 50%. The bar indicates the number of changes per length of branch. Species with verrucae marked (*), species with acrorhagi marked (+), species with vesicles marked (&), species with pseudoacrorhagi marked (^).

Figure 5: Comparison of results of Geller and Walton (2001) analysis compared to current analysis. A) Strict consensus tree of 25 most parsimonious trees from sequences from Geller and Walton (2001) and current analysis. Sequences from Geller and Walton’s 2001 analysis of Anthopleura were downloaded from GenBank and compared with sequences generated from this analysis. The symbol + indicates sequences from Geller and Walton, 2001. B) The best maximum likelihood tree with bootstrap values written on it. Bootstrap values are shown when they support a clade by more than 50%. Geller and Walton (2001) sequences were downloaded from GenBank. The symbol + indicates sequences from Geller and Walton, 2001. Bar indicates how many changes per length of branch.