ITS2 (Internal Transcribed Spacer2: rDNA) evolution and phylogenetics in Octocorals (Cnidaria: Anthozoa)

Daniel Guillermo Dorado Navarrete

A thesis submitted in partial fulfillment of the requirements for the degree of

Master of Science

Advisor JUAN ARMANDO SANCHEZ Marine Biologist Ph. D

Co-Advisor JENNY DUSSAN Microbiologist Ph. D

UNIVERSIDAD DE LOS ANDES FACULTAD DE CIENCIAS DEPARTAMENTO DE CIENCIAS BIOLOGICAS BOGOTA 2007

ITS2 (Internal Transcribed Spacer2:rDNA) evolution and its phylogenetics in Octocorals

RESUMEN Los octocorales gorgonaceos se encuentran dist ribuidos alrededor del mundo y en los arrecifes coralinos del Caribe forman jardines de enorme importancia ecológica. Sin embargo, las relaciones filogenét icas entre los distint os grupos no es clara debido a la ambigüedad de algunos caracteres morfológicos utilizados. Por tal razón, el uso de marcadores moleculares se ha convertido en herramienta esencial para establecer las relaciones en este grupo de organismos. En trabajos previos se demostró la utilidad del ITS2 (Internal Transcribed Spacer 2 : ADNr) como marcador molecular en octocorales, pero al mismo tiempo se evidencío su extrema variabilidad en todos los niveles (Intragenómico, Intraespecífico e Interespecífico), lo cual fue observado en patrones de DGGE con múltiples bandas por muestra; esta variabilidad no era esperada dado que se ha reportado que la evolución en concierto actúa sobre familias multigenicas (como el ADNr) causando su homogenización. Múltiples hipótesis han sido propuestas para explicar esta variabilidad, incluyendo la presencia de pseudogenes, variabilidad alélica, presencia de especies crípt icas y fenómenos de hibridación.

En este trabajo fueron realizados análisis más profundos para hallar las razones de la hipervariabilidad del ITS2, para lo cual fueron procesadas 114 muestras de 24 especies de octocorales pertenecientes a los géneros Eunicea, Pseudopterogorgia y Gorgonia, el IT S2 amplificado y corrido en DGGE. Las bandas observadas en el DGGE fueron cortadas y reamplificadas para su secuenciación. Las secuencias fueron usadas para la predicción de sus estructuras secundarias, permitiendo que fuese creada una matriz con caracteres morfológicos de las diferentes estructuras. Con los resultados obtenidos, por primera vez se demuestra la presencia de pseudogenes en el IT S2 de oct ocorales y su cont ribución en la diversidad de est a región. También fueron hallados múltiples alelos (probablemente funcionales) como fuente de diversidad intragenómica, intraespecífica e intragénerica del ITS2. Como resultado inesperado fueron encontrados dos alelos compartidos entre algunas especies de las familias Plexauridae y Gorgoniidae, inicialmente fueron descartados problemas de contaminación por las variaciones de estos alelos, y luego se descartó la hipótesis de que estas copias fueran remanentes evolutivos de un ancestro común dada la gran conservación de sus estructuras secundarias. Por lo cual, se plantea la posibilidad de que estas especies estén compartiendo alelos funcionales.

La información obtenida a partir de las secuencias primarias y la matriz de los caracteres morfológicos de 16 especies de octocorales y 21 secuencias obtenidas del Genbank, fue usada además para realizar análisis filogenéticos de las familias Plexauridae y Gorgoniidae. El análisis de las secuencias se realizó por medio de Máxima Parsimonia (MP), Máxima verosimilitud (ML) e Inferencia Bayesiana (BI). El análisis combinado de las secuencias con los caracteres morfológicos de las est ruct uras secundarias fue realizado por medio de Máxima Parsimonia. Los result ados obtenidos corroboran que ninguna de las dos familias estudiadas son monofiléticas, lo cual fue observado en clusters con soporte fuerte (Bootstrap y probabilidad posterior) que agrupaban especies de ambas familias como en el caso del cluster Muriceopsis-Pterogorgia previamente reportado.

En trabajos anteriores fueron descritos tres morfotipos distintos de Pseudopterogorgia bipinnata a diferent es profundidades, los cambios morfológicos fueron explicados como plast icidad fenotípica frente a distintas condiciones ambientales y no por variaciones genéticas. En este estudio fueron incluidas varias muest ras de cada uno de los morfot ipos, obt eniendo como result ado una gran homogeneidad en las secuencias y por ende en sus estructuras secundarias. En todos los análisis filogenéticos estas muestras se agrupaban sin ninguna distinción morfológica, por lo cual se corrobora los resultados descritos anteriormente.

TAB LE O F CO NTENTS

LIST OF FIGURES...... 5

CHAPTER I: Pseudogenes and allele variation contribute to the extreme variability of ITS2 (Internal Transcribed Spacer 2, rDNA) in octocorals (Cnidaria: Anthozoa)

Abstract...... 6 Introduction...... 6 Materials and Methods...... 8 Results...... 10 Discussion...... 13 Acknowledgements...... 21 References...... 21

CHAPTER II: ITS2 (Internal Transcribed spacer 2: rDNA) and its predicted secondary structure as molecular tool for the reconstruction of phylogenetic hypotheses of Caribbean shallow-water octocorals (Cnidaria: Anthozoa)

Abstract...... 25 Introduction...... 25 Materials and Methods...... 27 Results...... 31 Discussion...... 38 Acknowledgements...... 43 References...... 43

Appendixes ...... 46

LIST OF FIGURES

Figure 1.1. DGGE (Denaturant Gradient Gel Electrophoresis) analyses. a. Gorgonia species showing single and multiple band patterns. Similar band patterns from close locations and dissimilar from distant locations also seen in b. with E. succinea and E. palmeri. Blue arrows indicate bands cut from the gel. Green arrows indicate actual sequenced bands...... 11

Figure 1.2. P. bipinnata ITS2 alleles predicted secondary structure. ∆G, location and length are included for each case. a. General features of the octocorals secondary structure model and the morphometric characters used are indicated...... 14

Figure 1.3. Comparison of alleles vs. pseudogenes from the same genomes, the characteristics that were important for the determination of pseudogenes are shown: ∆G, length of the sequence, predicted secondary structure and pairwise alignment with consensus sequence. a. G. flabellum b. E. succinea...... 16

Figure 1.4.. Examples of conserved predicted secondary structures trough different levels, ∆G, length of the sequence and sample collection location are included. Arrows indicate the homologous structures a. Intragenomic allele variation. b. Intraspecific allele variation. c. Intrageneric ITS2 structure conservation. d. Intrafamiliar ITS2 structure conservation...... 18

Figure 1.5. Common alleles (a and b) found to be shared by Plexauridae and Gorgoniidae species, ∆G, length of the sequence and sample collection location are included...... 19

Figure 2.1. Predicted secondary structure of a sample of P. bipinnata, the structure corresponds to the conserved six- helicoidal ring-model. The characters used for the construction of the morphological matrix are shown: (N: Number, L: Letter) N stem, N` opposite side stem, Ni terminal loop, i` spacer, NL internal loop, N`L opposite side of the internal loop...... 28

Figure 2.2. Examples of ITS2 predicted secondary structures of different species. Is included ∆G and geographical location where the sample was taken...... 30

Figure 2.3. Bayesian inference of phylogeny of all samples including GenBank sequence (only sequences). Inference was done using MrBayes according to the model SYM+I+G (hTLR) suggested by MrModeltest with 1000000 generations, sample frequency of 1000 and burning zone of 500. Blue samples correspond to Gorgoniidae family, Black samples correspond to Plexauridae family and green were Alcyonacean octocorals as outgroups. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families. Posterior probability is also shown...... 32

Figure 2.4. Maximum Likelihood Hypothesis of all samples including GenBank sequence (only sequences). Inference was done using PAUP according to the model GTR+G+I (AIC) suggested by Modeltest, Majority rule (50%). Bootstrap 100. Blue samples correspond to Gorgoniidae family, Black samples correspond to Plexauridae family and green were Alcyonacean octocorals as outgroups. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families. Bootstrap is also shown...... 34

Figure 2.5. Maximum Parsimony combined hypothesis of primary and morphological characters. Inference was done using PAUP, Majority rule (50%). Bootstrap 1000. 279 parsimony-informative characters from 370. Blue samples correspond to Gorgoniidae family, Black samples correspond to Plexauridae family and green were Alcyonacean octocorals as outgroups. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families, conserved secondary structure is also shown. Bootstrap results are shown...... 37

Figure 2.6. (Previous page) Maximum Parsimony Combined hypothesis of primary and morphological characters for Gorgoniidae (a.) and Plexauridae (b) families. Inference was done using PAUP, Majority rule (50%). Bootstrap 1000. Blue samples correspond to Gorgoniidae family and Black samples correspond to Plexauridae family. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families. Bootstrap results are shown. a. Gorgoniidae tree. 225 parsimony-informative characters from 337. b. Plexauridae tree. 268 parsimony-informative characters from 374...... 40

Pseudogenes and allele variation contribute to the extreme variability of ITS2 (Internal Transcribed Spacer 2, rDNA) in octocorals (Cnidaria: Anthozoa)

Abstract Previous studies demonstrate the high ITS2 (Internal Transcribed Spacer 2: rDNA) diversity in octocorals at all levels (Intragenomic, intraspecific and interspecific) observed as DGGE complex band patterns. In this study further analyses were done in gorgonian octocorals to find the causes of the ITS2 variability, to achieve this, DGGE bands were excised, sequenced and its RNA secondary structure predicted. Results obtained demonstrated the presence of ITS2 pseudogenes for the first time in octocorals and its contribution for ITS2 diversity. In addition, multiple alleles were described as a source of ITS2 intragenomic, intraspecific and intrageneric diversity. As unexpected result two conserved alleles were shared by both, Plexauridae and Gorgoniidae families, to explain this, hypothesis of contamination and remnants ITS2 copies from common ance stor we re discarde d and thus functi onal share d allele hypothesis was proposed.

Introduction Octocorals are among the most important shallow and deep-water colonial invertebrates, comprising a large and diverse group in the Caribbean with more than 60 species in single coral reefs (Sanchez 1999), characterized by the presence of eight pinnate tentacles around the polyp mouth and a flexible colonial mass composed of fused sclerit es unit ed by a fleshy coenenchyme and in some cases a scleroprotein skeleton is present (Cairns 1976). Unlike hard corals (Scleractinean) the identification of octocorals is extremely difficult and ambiguous, and requires in most cases the examinat ion of sclerit es through the microscope, imposing a difficulty in their study, resulting in a small number of studies in this group compared to hard corals (Sanchez & Wirshing 2005), this has lead the use of molecular markers to fill the gap of morphological ambiguities.

Recently, many molecular markers have been used in octocorals as mitochondrial rDNA 16s and nuclear rDNA 18s (Bridge et al. 1995; France et al. 1996; Won et al. 2001; Berston et al. 2001; Sanchez et al. 2003), mitochondrial NADH dehydrogenase subunit s (ND2, ND3 and ND6), mitochondrial COI and COII spacers, mitochondrial mutS-like MSH1 gene (France & Hoover 2001; McFadden et al. 2002; Sánchez et al. 2003; Wirshing et al. 2005; McFadden et al. 2006) and microsat ellit es (Gut ierrez-Rodriguez & Lasker, 2004; Liu et al. 2005a; Liu et al. 2005b; Baco et al. 2006). However, most nuclear and mitochondrial molecular markers have shown a great deal of conservat ion among closely-relat ed species (Romano & Palumbi 1997, Sanchez et al. 2007). To solve this problem, Internal Transcribed Spacers located in the rDNA have been used as molecular markers because their size, sequence and complexity vary considerably among species (Odorico & Miller 1997). In octocorals, ITS2 have been used to resolve relations between closely related species within genera and family (MacFadden et al. 2001, McFadden & Hutchinson 2004; Aguilar & Sánchez, 2007, Grajales et al. 2007).

rDNA (ribosomal DNA) is a multigene family arranged in tandem repeats separated by Intergenic Spacers (IGS), frequently achieving several hundreds of repetitions per chromosome. Each repet it ion is composed by three ribosomal subunit s (18s, 5,8s and 28s) separated by two internal transcribed spacers (ITS1 and ITS2) (Cote & Peculis 2001). ITS1 and ITS2 spacers form secondary structures crucial for ribosomal maturation, which are know to have conserved core structure through metazoans, important for the maturation of the rRNA. Changes in this struct ure are know to produce inhibit ion of the mat urat ion of rRNA due to co-evolution between RNA secondary structures and the processing molecular machinery responsible for its removal (Van Nues et al. 1995).

As multigene family, the rDNA is suppose to evolve via concerted evolution result ing in the homogenizat ion of the sequences throughout the genome (Harris & Crandall 2000, Hillis & Davies 1988) i.e., homogenization of copies through unequal crossing over and gene conversion (Li 1997). However, variations within individuals have been reported primarily due to slower concert ed evolut ion (Harris & Crandall 2000, Cot e & Peculis 2001), hybridization or by the presence of pseudogenes (Marquez et al. 2003, Harpke & Peterson 2006). The former probably due to the presence of highly divergent rDNA types in different chromosomes (Arnheim et al. 1980) resulting in the maintenance of ancestral rDNA polymorphisms for long times (Marquez et al. 2003), on the other side, hybridizat ion phenomena between species per se could increase the rDNA diversity in an individual, but as additional consequence could result in silencing of some rDNA loci by chromat in modificat ions in a nucleolar dominance process (chen et al 1998, Frieman et al. 1999, Muir et al. 2001) causing that some rDNA lo ci could be driven by neutral select ion as pseudogenes (Muir et al. 2001).

7

Recently, a study that validated the ITS2 reliability as molecular marker for fingerprinting in Octocorals also demonstrated variations in ITS2 at intragenomic, intraspecific and interspecific level as multiple bands in DGGE analyses, which was hypothesized to be probably due to allele variation, incomplete lineage sorting, cryptic species, heteroduplex DNA and/or pseudogenes (Dorado 2006). In this study further analyses were done in 23 species of genus Eunicea, Gorgonia and Pseudopterogorgia to understand the variation of ITS2 previously demonstrated in Octocorals. To achieve this, DGGE analyses were done and bands observed in the patterns were cut, sequenced and its secondary structure predicted. Based on this information, pseudogenes and allele variations were searched trough all the samples studied as possible explanations for ITS2 diversity.

Mate rials and Methods Sample collection, identification and DNA extraction. Octocoral samples were collect ed during May 2005 - Oct ober 2006 from six different locations between Cartagena and Rosario Islands using SCUBA diving. 80 samples from 25 species with emphasis in the genera Eunicea, Gorgonia and Pseudopterogorgia were collect ed and preserved in absolute ethanol. Samples from other Caribbean locations were obtained from Biommar and Dr. Sanchez collect ions which were t agged and organized for this study (Refer to appendix A and B for sample details). All collected samples were identified trough microscope comparison of sclerit es using the guide by Bayer (1961). Total DNA was extracted from 235 samples of 30 species using a CT AB, prot einase K, Phenol – Clorophorm-IsoAmyl alcohol extraction method (Coffroth et al. 1992), DNA was resuspended and conserved in TE buffer at – 70ºC. DNA quality was checked in agarose (1%) electrophoresis at 80V for 30 min. 27 DNA samples of Pseudopterogorgia bipinnata from previous st udies were also included in this work (Sanchez et al. 2007).

PCR A total of 177 DNA extraction samples of 29 species were chosen, including at least 6 samples per species that showed the best DNA extraction quality. Primers 5.8s 5` AGCAT GT CT GT CT GAGT GTT GG 3 ` an d 28 s 5 `GGGT AAT CTT GCCT GAT CT GAG3 ` designed by Aguilar and Sanchez (2007) were used for the IT S2 amplificat ion, condit ions for PCR were as follows: an initial denaturizing step: 2 min at 94ºC, followed by 35 cycles: 30 sec. at 94ºC, 30 sec. at 56.8ºC and 1 min at 72ºC, final extension step: 2 min. at 72ºC. Using 1 unit of Taq polymerase (Invitrogen), 3.5 mM MgCl2, 0.2 mM DNTPs (Biorad Mix), 0.15 uM primers (each) and 4µL of DNA (dilution 1/50) in 20µL as final volume. The amplification

8 was standardized with an efficiency of 95% in Pseudopterogorgia and Go rg on ia samples, and 55% in Eunicea Samples (Refer to Appendix A for PCR efficiency details).

DGGE, Band extraction and DNA precipitation 114 PCR react ions of 23 species were separat ed in a gel cont aining 8% polyacrilamide, 1X TAE Buffer and linear urea-formamide denat uring gradient from 45% to 80%. The gels were pre-run at 60ºC and 90V for 30 min, followed by the electrophoresis at 60ºC and 90V for 13 h. Gels were st ained wit h Et hidium Bromide during 15 min and visualized using a BIORAD chemidoc system. All reactions were conducted without CG- clamp. Bands visualized in the DGGE gel were cut using sterilized micropipette tips in the BIORAD chemidoc system and placed in 0,5 mL tubes with 100µL of sterilized double distilled water. The tubes were incubated in a shaker at room temperature for 24 hours at 150 rpm. Each band extract was collected in a 0,5 mL tube and the DNA was precipitated with 300 µL of cold absolute Ethanol, tubes were placed at -20ºC for 24 hours and then centrifuged at 13000 rpm for 30 min, supernat ant was discarded and pellet dried and resuspended in 15 µL sterilized double distilled water.

ITS2 sequences PCR of DNA Bands was conducted as described above except that DNA was used wit hout dilut ion. Purificat ion of PCR product s for sequencing was performed by Exo-Sap method using 1u of Exonuclease, 0,2 u of Shrimp Alkaline Phosphatase and 2µL SAP buffer 10x per 20 µL in a 0,2 mL tube. Reactions were held at 37ºC for 1 hour and at 80ºC for 15 min. Sequencing reactions were performed with BigDye 3.1 system according to manufactures instructions (Applied Biosystems) and sequenced in a capillary electrophoresis automated sequencer (ABI3100). Each sample was sequenced with forward and reverse primers, the consensus sequences were obtained by assembling the two complementary chromatograms in Sequencer 4.7 software. 42 ITS2 sequences of Octocorals were also obtained from GenBank (refer to appendix B for samples details).

Prediction of ITS2 RNA secondary structures Secondary structures of all sequences obtained (including GenBank sequences) were reconstructed by comparison via Pairwise Alignment (Bioedit) with previously reported structures in Octocorals (Aguilar & Sanchez 2007). The sequences were then submitted with constrains and restrict ions in MFOLD (Zuker, 2003) at default temperat ure of 37ºC, the structure chosen was the one with greater negative free energy but with the ring model more similar to the reported. The obtained secondary structures were used to construct a matrix for

9 cladistic analysis as described by Aguilar & Sanchez (2007). Number of nucleotides for each structure character were counted and arranged in discrete character states ranging from 0 to 9, were 0 is absence of the character and 1 the less nucleotide number (See Appendix E and F).

Pseudogenes determination Pseudogenes were det ermined trough a comparison of intragenomic secondary structures (when multiple sequences per sample were available) and/or intraspecific secondary structures based on previously reported works (Marquez et al. 2003). Sequences were pseudogenes candidat es if ∆G of the secondary structure, length of the sequence and structural features were considerably different from other secondary structures of the sample and/or species. Pairwise alignments of the possible pseudogenes with a consensus structure of the sample or species were done to find indels or other causes of the differences. Only sequences with extreme variations and with proved causes of the differences were considered as pseudogenes.

Results Amplification was successful in Pseudopterogorgia and Go rg on ia species. However the genus Eunicea presented lower amplification efficiency, probably due to pigments that were not separated, even when Phenol-Clorophorm steps were duplicated. Species with amplification problems were E. sp. 2, E. tourneforti and E. knighti all with high level of pigment at ion. On the cont rary Eunicea species with low level of pigmentation as E. pallida were successfully amplified (Refer to appendix A for PCR efficiency details).

DGGE All species showed multiple banding patterns at least in one sample, however some samples of Gorgonia ventalina, G. mariae, and Pseudopterogorgia bipinnata showed single banding patterns (fig. 1.1). Multiple banding patterns ranged from 2 to 7 bands per sample. Samples of the same species from closer geographical location showed similar patterns with shared bands but with differences in bands intensity (e.g. Gorgonia and Eunicea species in figure 1.1), differences were obvious between samples from distant locations with exception of E. mammosa and P. acerosa where all patterns no matter the locations showed common bands (results not shown). In Eunicea and Pseudopterogorgia species the degree of intraspecific variation was high with little resemblance between samples of the same species. In other cases different bands could be shared by different species (i.e. E. succinea and E. palmeri in fig. 1.1). In this study all banding patterns found in DGGE were replicable, demonstrating the usefulness and reliability of this technique.

10

a

b

Fig. 1.1 DGGE (Denaturant Gradient Gel Electrophoresis) analyses. a. Gorgonia species showing single and multiple band patterns. Similar banding patterns from nearby locations and dissimilar from distant locations also seen in b. with E. succinea and E. palmeri. Blue arrows indicate bands cut from the gel. Green arrows indicate actual sequenced bands.

11 ITS2 Sequence Only sequences that had in both extremes the primer sequence (at least partially) were used. Extreme variability in ITS2 length was obtained, ranging form 154 bp to 281 bp with an average of 236 bp, unexpect edly bot h short er and longer amplificat ions product s were obtained from different DGGE bands of a sample of Eunicea succinea (a complete reference of length of IT S2 of all samples and species used in this study including GenBank Sequences is included appendix C and D). The genus Eunicea presented an average ITS2 length of 236 bp with great variability and genus Pseudopterogorgia presented an average length of 223 bp. Great differences in length of different intragenomic copies were the first indication of the presence of multiple gene copies.

Secondary structure Predicted secondary structures followed the conserved six-helicoidal ring-model previously reported for octocorals (Aguilar & Sanchez 2006) (see fig. 1.2), but great variability was observed in the length and complexity of each stem and spacer. In general the stems II, III and VI are shorter than the stems IV and V, being the stem V the longest. Multiple internal loops are frequent in stems III, IV and V, with major number in stem V where were observed up to six internal loops. The spacers were more often short ranging from 1 to 4 bp, the spacer 1i showed a conserved sequence UG with little variations across the studied species, the spacer 4i was the longest with 4 to 12 bp with a conserved core sequenced AGUNCAGC observed in most of the studied samples.

Intragenomic differences were frequently discrete changes that did not affect the predicted secondary structures (fig. 1.2) but in some occasions differences were enough to dramatically change ITS2 structures, even causing disappearing of the six-helicoidal ring model (fig. 1.3a and 1.3b). The former changes were considered as allele variation while severe changes were considered as pseudogenes. Two sequences from G. flabellum and E. succinea were finally considered as unquest ionable pseudogenes, due to the loss of characteristics present in all other secondary structures of the same sample and for evident delet ions of ent ire fragment s up to 100 bp seen in the alignment s of the sequences (fig. 1.3a and 1.3b). Ot her four sequences from Eunicea species were considered as possible pseudogenes (fig. 1.3c) for evident changes in the struct ure, lower ∆G and length of the sequence, but the alignment s did not give enough informat ion to explain the changes.

Variable results were obtained concerning allele diversity according to the taxonomic level analyzed and the geographical location. Multiple functional alleles with conserved

12 structural features at intragenomic and intraspecific level were more frequently found in samples that came from close geographical locations than from distant locations (i.e. fig. 1.5), however, examples of conserved alleles from distant places were found (i.e. E. succinea and E. flexuosa, data not shown), wh ich was co n gruent wit h DGGE result s (i.e. P. bipinnata). Conservation of structural features was also observed at genus, and family level (fig 1.5), and surprisingly two conserved alleles were found between Plexauridae and Gorgoniidae species (fig 1.6).

Discussion Results of DGGE were consistent with sequences analyses and secondary structure prediction, samples of the same species from nearby locations had similar patterns as seen in predicted secondary structures, and in the opposite situation samples from distant locations showed dissimilar banding patterns and secondary structures (i.e. P. bipinnata and E. pallida, data not shown). T his suggest ed barriers that reduce the reproduct ive possibilities between individuals from dist ant locat ions, meaning that all the IT S2 genet ic pool of some octocoral species are not necessarily equally geographically dist ribut ed, and thus some alleles could be more frequent of even absent depending the locat ion.

Bands were cut from DGGE gels with the assumption that same level bands were the same sequence (or very similar). However to test that assumption bands at the same level from different specimens (P. rigida, P bipinnata) were cut and sequenced, but the struct ures were no exact ly the same (Dat a not shown). Thus is recommended for upcoming studies not to assume that bands at same level in DGGE have the same sequence.

Secondary structure prediction Although all the main structures were always present their variation in length, sequence and shape was remarkable. Only some conserved features were found (see results), indicating a strong selection on these conserved structures, probably needed by the molecular machinery for processing the ITS2 as was demonstrated by Van Nues et al. (1995).

Alt hough secondary struct ures were useful and provided addit ional phylogenet ic and ITS2 evolutive information, it was clear that the predicted secondary structures can not be considered the true structures (Harpke & Peterson, 2006), because the prediction is done through heavily manipulable methods. In this concern, some of the predicted structures done in t his work were different that those proposed previously from the same sequences (Aguilar & Sanchez 2007) and probably upcoming works will predict multiple different structures from a

13 single sequence, thus is considered that there is a need for methods as X-Ray Crystallography to solve the real structure of ITS2 in octocorals.

a.

Fig. 1.2 P. bipinnata ITS2 alleles predicted secondary structure. ∆G, location and size are included for each case. a. General features of the octocorals secondary structure model and the morphometric characters used are indicated.

Pseudogenes as contribution for extreme ITS2 diversity in octocorals The rDNA as a multigene family is supposed to be homogenized by concerted evolution (Harris & Crandall 2000), but mult iple copies per genome could be found as has been reported in different groups of organisms (Odorico & Miller 1997, Hugall et al. 1999, Harpke & Peterson 2006). This is true for octocorals where multiple ITS2 copy were reported in a single genome as complex banding patterns in DGGE analyses (Dorado 2006), one of the

14 proved reasons in other organisms to explain this variability is the presence of pseudogenes (Marquez et al. 2003). In this work for the first time pseudogenes of ITS2 were reported in oct ocorals, two pseudogenes were found which had evident delet ions (46 and more than 100 bp) compared with what we consider normal alleles (see fig. 1.3), these deletions caused the lost (total or partial) of the stem V, which is predicted to be necessary for the maturation of rRNA as a conserved structure in all octocorals (Aguilar & Sanchez 2007). Other four ITS2 copies from Eunicea species were found to posses one or more pseudogenes charact eristics compared with consensus sequences as differences in length of the sequence, ∆G considerable higher (less negative) and important structural changes. These characteristics probably affect the ability of these sequences to be effectively spliced to form the ribosome subunit s due t o the known specificity and little tolerance of changes of the splicing responsible machinery to the ITS2 secondary structure (Van Nues et al. 1995), thus is predicted that only alleles with minor changes could be functional. Interestingly these four sequences were placed together in a single cluster in the phylogenetic analyses even when they belong to different Eunicea species (see chapter 2), what could be an indication that this sequence could be a remnant of a former ITS2 sequence of the common ancestor and it is still carried by derived species or the effect of long branch att ract ion . This hypot hesis should be t ested in further st udies focused on Eunicea ITS2 evolution.

Other copies of ITS2 present in the genome that showed less abrupt changes or even some of the predicted funct ional alleles could be pseudogenes (or even less efficient but functional sequences), but the methodology used in this work do not allow to detect them. Thus, it is recommended using of methodologies as RT-PCR for the detection of the ITS2 alleles being silenced (Marquez et al. 2003) and in-vivo works to prove the biological and expression consequences of each small or big change (if are allele variation or pseudogenes), proving the real contribution of pseudogenes in the variability of ITS2 (Van Nues et al. 1995). It is clear that pseudogenes contribute to the diversity of ITS2 in octocorals (i.e. E. succinea sample oct32 where pseudogenes brings 60% of the diversity) but it is most likely that the det ect ed cont ribut ion is an underestimat ion of the real one.

The results obtained neither confirm nor discard hybridization phenomena in the species studied, and is clear that further studies should sequence all the bands present in every sample to confirm this hypothesis. However, the hybridization phenomena is known to happened in some octocorals (McFadden & Hutchinson 2004) which would contribut e in the diversity of IT S2 in individuals, not only for the parent al alleles cont ribut ion but also by the nucleolar dominance phenomena where one of the parental chromosomes could be silenced

15 partially or totally (Chen et al 1998), generating the evolution of the silenced loci in a neutral fashion. The silenced regions are frequently methylated especially in the cytosine, and the methylcytosine residues spontaneously tend to deaminate resulting in mutations to thymine (Marquez et al. 2003). Thus, pseudogenes caused by this phenomena could have a higher proport ion of AT than funct ional alleles do. So, is recommended in upcoming works to test the presence of pseudogenes wit h the addit ional CG – AT proport ion comparison crit erion, as previously proposed by Marquez et al. (2003).

a

b

Fig. 1. 3 Two unquestionable pseudogenes found: comparison of alleles vs. pseudogenes from the same genome. The characteristics that were important for the determination of pseudogenes are shown: ∆G, length of the sequence, predicted secondary structure and pairwise alignment with consensus sequence. a. G. flabellum and b. E. succinea.

Alleles as contribution for extreme ITS2 diversity in octocorals It is clear that multiple copies of ITS2 with similar characteristics were present in a single genome (fig. 1.4a), these copies were probable functional alleles and its small variations contribute to the extreme diversity without affecting its function (Odorico & Miller 1997). In some samples (i.e. P. bipinnata), these allele variations explained the total ITS2 diversity observed (fig. 1.2). It was also remarkable the contribution of functional allele variability in the intraspecific ITS2 diversity, which in a geographical location could be responsible for the entire diversity found, as seen in P. bipinnata in Belize and Panama (fig. 1.2). As expected,

16 more intraspecific differences in the characteristics of ITS2 copies were observed in distant locations, due probably to geographical barriers that limit the genetic flow or to incomplete lineage sorting, however further genetic population studies with other molecular markers as microsatellites should be performed to understand the genetic flow in Caribbean octocorals.

Some species samples (i.e. E. flexuosa) from distant locations showed similar IT S2 structural characteristics (Data not shown), indication of conserved intraspecific machinery for rRNA maturation. On contrary, two samples of P. bipinnata from close locations (Panama and Colombia) presented different structural features (fig. 2), this is unexpected because significant changes in intraspecific structures probably means major changes in molecular splicing machinery (Van Nues et al. 1995), which is unlikely at species level, a comfortable explanation for these changes could be the presence of cryptic species (Bickford et al 2006) wit h different IT S2 struct ural feat ures or even a wrong ident ificat ion of the species, however the only source of these sample was dried DNA from previous works thus a morphology and sclerites analyses needed to test this hypothesis were not possible. However, it is also possible t hat diversit y wit hin and among species wit h recent divergence could be explained by incomplete lineage sorting (Van Oppen et al. 2001), seen as incomplete fixation of alleles in all the species populations.

Intrageneric conserved structural ITS2 features (see fig. 1.5 c) could be explained as the result of the common history of close related species and thus the resemblance of the rRNA mat urat ion machinery and the IT S2 struct ures, or could be t he result of a hybridizat ion phenomenon which was previously reported in other groups of organisms (Harpke & Peterson 2006). The former explanation is more likely, but analyses with all bands should be performed to support it. At the intergenus level, conserved structures were seen between Plexaura homomalla and Pseudoplexaura crucis (fig 1.5d) and unexpectedly the resemblance of the P. homomalla was longer to P. crucis than to Plexaura kuna, indicat ion that a revision of the systematic of these species should be made.

The most unexpected result is the presence of conserved structures of two alleles between Plexauridae and Gorgoniidae species, the first one was found between Gorgonia spp. and Eunicea pallida (fig 1.6a) and the second between Pseudopterogorgia spp. and Eunicea spp. (fig. 1.6b), each of these alleles are presented at least in seven samples. At first sight it was believed t hat a cross cont aminat ion was possible, however differences bet ween the copies and the high number of samples did not support this explanation, additionally, samples of the species involved never were processed together and even some sequences comes from

17 GenBank. The second possibility was that these structures were evolutive remnants from a common ancestor, but it was rejected due to high conservation of the structure which is not suppose to happened if the sequence is not longer functional and driven by neutral selection (Newman & Engelhard 1998). Thus, one possible explanation is that these conserved sequences are drove by a strong selection which would be responsible for the maintenance of the structure but means that these are functional ITS2 sequences. Other important element for this analysis is that more bands at the same level to the conserved copies were seen in DGGE gels but were not sequenced for limit at ions in resources and time as explained above, meaning that these copies could be more frequent and even present in more species. This is a hypothesis that should be tested very carefully because no previous work supports it and for the implications that it will carried.

18 Fig. 1.4 Examples of conserved predicted secondary structures trough different levels, ∆G, length of the sequence and sample collection location are included. Arrows indicate the homologous structures a. Intragenomic allele variation. b. Intraspecific allele variation. c. Intrageneric ITS2 structure conservation. d. Intrafamiliar ITS2 structure conservation.

19

Fig. 1.5 Common alleles (a and b) found to be shared by Plexauridae and Gorgoniidae species, ∆G, length of the sequence and sample collection location are included.

20

In general it was clear that concerted evolution do not effectively homogenized ITS2 sequences in oct ocorals, and that pseudogenes and allele variat ions are major diversit y factors for ITS2. The inefficient concerted evolution effect over rDNA homogenization, could be produced by the location in the genome of the different ITS2 alleles, because is know that homogenizat ion could be slow down if t he different alleles are locat ed in different chromosomes, due to lower gene conversion and unequal crossing over bet ween not homologous chromosomes (Arnheim et al. 1980). Other possible causes for ITS2 diversity as hybridization, cryptic species and incomplete lineage sorting were not proved but stills remain as feasible.

Acknowledgements This st udy was part ially funded by COLCIENCIAS (No. 120409-16825) and the Facultad de Ciencias, Department of Biological Sciences, Universidad de los Andes (funding to J.A. Sánchez). We are grateful with BIOMMAR divers and colleagues for their assistant during field surveys and laborat ory assays.

References Aguilar, C., Sánchez, J.A. (2007) Phylogenetic hypothesis of gorgoniid octocorals according to ITS2 and their predicted RNA secondary structures. Mol. Phylogenet. Evol. 43:774-786.

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21 Bridge, D., Cunningham, C.W., DeSalle, R., Buss, L.W. (1995) Class-Level Relationships in the Phylum Cnidaria: Molecular and Morphological Evidence. Molecular Biology & Evolution 12, 679-689 Cairns, S. (1976) Guide to the common shallow-water gorgonians of Florida, the Gulf of Mexico, and the Caribbean region. Sea grant Field Guide Series, 6, University of Miami, Miami, FL. Chen, Z.J., Comai, L., Pikaard, C.S. (1998) Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids. Proc. Natl. Acad. Sci. USA 95:14891-14896. Coffroth, M.A., Lasker, H.R., Diamond, M.E., Bruenn, J.A., Bermingham. E. (1992) DNA fingerprint s of a gorgonian coral: A method for det ect ing clonal struct ure in a veget at ive species. Marine Biology 114, 317-325. Coté, C.A., Peculis, B.A. (2001) Role of the ITS2-proximal stem and evidence for indirect recognition of processing sites in pre-rRNA processing in yeast. Nucleic Acids Research 29, 2106-2116 Dorado, D. (2006) Uso del Espaciador Trascrito Interno 2 (ADNr) como marcador de especies de octocorales. Universidad de los Andes, Biology Thesis 2006. France, S.C., Rosel, P.E., Agenbroad, J.E., Mullineaux, L.S., Kocher, T.D. (1996) DNA sequence variat ion of mitochondrial large-subunit rRNA provides support for a two-subclass organization of the Anthozoa (Cnidaria). Molecular Marine Biology & Biotechnology 5, 15- 28. France, S.C., Hoover, L.L. (2001) Analysis of variation in mitochondrial DNA sequences (ND3, ND4L, MSH) among octocorals (=Alcyonaria) (Cnidaria: Anthozoa). Bulletin of the Biological Society of Washington 10, 110-118. Frieman, M., Chen, Z.J., Vasquez, J.S., Shen, L.A., Pikaard, C.S. (1999) RNA polymerase I t ranscript ion in a Brassica int erspecific hybrid and it s progenit ors: t est of transcript ion fact or involvement in nuclear dominance. Genetics 152:451-460. Grajales, A., Aguilar, C., Sanchez, J. (2007) Phylogenetic reconstruction using secondary structures of Internal Transcribed Spacer 2 (ITS2, rDNA): finding the molecular and morphological gap in Caribbean gorgonian corals. BMC Evolutionary Biology, 7:90 Gutiérrez-Rodríguez C, Lasker HR. (2004) Microsatellite variation reveals high levels of genetic variability and popµLation structure in the gorgonian coral Pseudopterogorgia elisabethae across the Bahamas. MolecµLar Ecology 13, 2211-21. Harpke, D., Peterson, A. (2006) Non-concerted ITS evolution in Mammillaria (Cactaceae) Molecular Phylogenetics and Evolution 41:573-593

22 Harris, D.J., Crandall, K.A. (2000) Intragenomic variation within ITS1 and ITS2 of freshwater crayfishes (Decapoda: Cambaridae): Implications for phylogenetic and microsatellite st udies. Molecular Biology & Evolution 17, 284-291. Hillis, D.M., Davis, S.K. (1988) Ribosomal DNA: interspecific polymorphism, concerted evolution, and phylogeny reconstruction. Systematic Zoology 37:63-66 Hugall, A., Stanton, J., Moritz, C. (1999) Reticulate Evolution and the Origins of Ribosomal Internal Transcribed Spacer Diversity in Apomictic Meloidogyne. Mol. Biol. Evol. 16(2):157-164. Li, W.H. (1997) Concerted evolution of multigene families. Molecular Evolution. Sinauer Assoc., Inc, Suderland, MA, pp. 309-334 Liu, S.Y.V., Dai, C.F., Fan, T.Y., Yu, H.T. (2005a) Genotyping the clonal structure of a gorgonian coral, Junceella juncea (Anthozoa: Octocorallia), using microsatellite loci. Coral Reefs 24, 352-358. Liu, S.Y.V., Yu, H.T., Fan, T.Y., Dai, C.F. (2005b) Cloning and Characterization of microsatellite loci in a gorgonian coral, Junceella juncea (Anthozoa ; Octocorallia ; Ellisellidae) and its application in clonal genotyping. Marine Biotechnology 7, 26-32. Marquez, L.M., Miller, D.J., Mackenzie, J.B., Van Oppens, M.J. (2003) Pseudogenes contribute to the extreme diversity of nuclear ribosomal DNA in the hard coral Acropora. Molecular Biology & Evolution 20, 1077-1086.

McFadden, C.S., Donahue, R., Hadlan, B.K., Weston, R. (2001) A molecular phylogenetic analysis of reproductive trait evolution in the soft coral genus Alcyonium. Evolution 55, 54-67. McFadden, C.S., Hutchinson, M.B. (2004) Molecular evidence for the hybrid origin of species in t he soft coral genus Alcyonium (Cnidaria: Anthozoa: Octocorallia). Molecular Ecology 13, 1495-1505. McFadden C. S., France, S. C., Sánchez, J.A. & Alderslade, P. (2006) A Molecular Phylogenetic Analysis of the Octocorallia (Cnidaria: Anthozoa) Based on Mitochondrial Prot ein-coding Sequences. Molecular Phylogenetics & Evolution 41, 3, 513-527. Muir, G., Fleming, C.C., Schlo, C. (2001) Three divergent rDNA clusters predate the species divergence in Quercus petrea (Matt.) Liebl. And Quercus robur L. Mol. BIol. Evol. 18:112-119.

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Molecular Species. RePEc: 98-01-001

23 Odorico, D.M., Miller, D.J. (1997) Variation in the ribosomal internal transcribed spacers and 5.8s ADNr among five species of Acropora (Cnidaria; Scleractinean): Patterns of variation Consistent with reticulate Evolution, Molecular Biology & Evolution 14(5): 465-473. Romano, S.L. and Palumbi, S.R. (1997) Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleract inean corals. J Mol Evol 45:397-411 Sanchez, J.A. (1999) Black coral – Octocoral distribution patterns on a deep-water reef, Imelda bank, Caribbean Sea, Colombia. Bull Mar Sci 65:215-225 Sanchez, J.A., McFadden, C.S., France, S.C. & Lasker, H.R. (2003) Molecular phylogenetic analyses of shallow-water Caribbean Octocorals. Marine Biology 142, 975-987. Sanchez, J.A., Wirshing, H.H. (2005) A field key to the identification of tropical western Atlantic zooxanthellate octocorals (Octocorallia: Cnidaria). Caribbean Journal of Science, 29, 508-522. Sanchez, J.A., Aguilar, C., Dorado, D., Manrique, N. (2007) Phenotypic plasticity and morphological integrat ion in a marine modular invertebrate. BMC Evolutionary Biology 7:122. Swofford, D. (2002) PAUP*, phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sunderland: SinauerAssociates. Van Nues, R. W., Rientjes, M.J., Morre, S.A., Molee, E., Planta, R.J., Venema, J., Raue, A.H. (1995) Evolutionary Conserved Elements are Critical for Processing of Internal Transcribed Spacer 2 from Saccharomyces cerevisiae Precursor Ribosomal RNA. J. Mol. Biol. 250, 24-36. Van Oppen, M.J., McDonald B.J., Wilis, B.L., Miller, D.J.. (2001) The evolutionary history of the coral genus Acropora (Scleractinea, Cnidaria) based on a mitochondrial and nuclear marker: reticulation, incomplete lineage soriting or morphological convergence? Mol. Biol. Evol. 18:1315-1329. Wirshing, H.H., Messing, C.G., Douady, C.J., Reed, J., Stanhope, M.J., Shivji, M.S. (2005) Molecular evidence for multiple lineages in the gorgonian family Plexauridae (Anthozoa: Octocorallia). Marine Biology 147, 497-508. Won, J., Rho, B., Song, J. (2001) A phylogenetic study of the Anthozoa (phylum Cnidaria) based on morphological and molecular characters. Coral Reefs 20, 39-50 Zuker, M., (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-3415.

24

ITS2 (Internal Transcribed spacer 2:rDNA) and its predicted secondary structure as molecular tool for the reconstruction of phylogenetic hypotheses of Caribbean shallow-water octocorals (Cnidaria:Anthozoa)

Abstract In Cari bbe an coral ree fs , gorgoni an octocorals cons tru ct con s pi cuous garde ns of enormous ecological importance. Most of the shallow water Caribbean octocorals are placed in the Plexauridae and Gorgoniidae families. However, it is no clear the relationships between and within these families and use of molecular markers has suggested that these families are not monophyletic. In this work, ITS2 was used as molecular marker for the reconstruction of a phylogenetic hypotheses of shallow water Caribbean octocorals, to achieve this, ITS2 of 16 species were sequenced, their secondary structure predicted and based on them a morphological matrix was done. Analyses of the primary sequences were done using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). The combination of primary characters and “morphological” characters from predicted secondary structures were done using MP analyses. Results obtained confirm that none of the families studied are monophyletic, seen as clusters with strong support formed by species from both families, as the Muriceopsis-Pterogorgia clus te r pre viou sly re porte d. In pre vious works th ree morphotypes of P. bipinnata where seen at different depths, it was reported that it was caused by phenotypic plasticity, results obtained in this work support this hypothesis due to low ITS2 variability between samples from different morphotypes seen as extremely conserved secondary structures and placement of all samples in a single cluster with no distinctions of morphotypes.

Introduction Octocorals are sessile marine invertebrates found from deep to shallow waters and from Artic to Antarctic latitudes (Bayer 1961). These organisms are cnidarians with only polyp state in all their life cycle (Class Anthozoa), and are characterized by the presence of eight tentacles per polyp (Sub-Class Octocorallia = Alcyonaria) (Sanchez & Wirshing 2005). The sub class Octocorallia is known to be a monophyletic group with great ecological importance with more than 3000 described species and a vast variety of niches occupied (France et al. 1996, Bernston et al. 2001). The classic phylogeny of these organisms is based on ext ernal and sclerit es morphology (Bayer 1961) which has lead very oft en to ambiguous result s due t o convergent evolut ion and high phenot ypic plast icity in mult iple charact ers used

25 in octocorals, this has lead the use of molecular markers to fill the gap of morphological ambiguit ies.

Gorgonian oct ocorals is a subgroup t hat is charact erized by the presence of a sclerit e- protein axis; however the order Gorgonacea (still in use in some databases) that grouped these organisms was abolished and included in the order Alcyonacea in the suborder Holaxonia (axis with hollow core) (Bayer, 1981) and Calcaxonian (axis with continuous core) (Grasshoff, 1999), but recent ly it was demonstrat ed that none of these suborders are monophylet ic according to molecular and morphological evidences (Sanchez et al. 2003a, McFadden et al. 2006). The Holaxonia suborder contains most of the shallow water gorgonian octocorals including Caribbean species that form dense gardens in coral reefs, most of them placed in the Plexauridae and Gorgoniidae families. The former is characterized by usual thick coenenchyme (connecting tissue) and large sclerites and Gorgoniidae by small sclerites and thinner branches (Bayer 1961). However characters used at the generic level are not clear and some species could be placed in both families (Sanchez et al. 2003b). It has been proven from multiple molecular markers (msh1, NADH subunit s 2 and 6 and ND2) and sclerit e EM, that none of these families are monophyletic (Sanchez et al. 2003b, Wirshing et al. 2005). Historically the most important genera from the shallow-water Caribbean octocorals included in the Plexauridae family are Pseudoplexaura, Plexaura, Plexaurella, Muricea, Muriceopisis and Eunicea and in Gorgoniidae family are Pseudopterogorgia, Pterogorgia, Gorgonia and Leptogorgia (Bayer 1961). However, recent work using mitochondrial molecular markers showed that genus Plexaurella branched at the base of Gorgoniidae family and genus Muriceopisis and Pterogorgia are grouped together without significant support within either families but closer with Gorgoniidae (Sanchez et al. 2003b, Wirshing et al. 2005). These findings should be tested using independent nuclear markers, which is a secondary objective of this work.

Recently nuclear and mitochondrial markers have been proposed to be useful for the phylogeny of octocorals (Sanchez et al. 2003a, McFadden et al. 2004) most of them valuable to solve relations between high level groups (Order, Suborder and families) but excessively conserved to solve closely-related species relationships (McFadden 2004). ITS2 spacer (Internal Transcribed Spacer 2) is the most variable region so far described in octocorals and t hus very good to solve close relat ionships, however some problems arise from it s use, as excessive INDELS and saturation (McFadden et al. 2004, Aguilar & Sanchez 2007). To overcome this problem the main biological characteristic of ITS2 was used, the prediction of its RNA secondary structure, which is know to have a major role in the maturation of

26 ribosomal RNA (Cote & Peculis 2001). The ITS2 predicted secondary structure in octocorals follow a conserved six-helicoidal ring-model (i.e. Aguilar & Sanchez 2007, Grajales et al. 2007, Sanchez et al. 2007), which provide useful “morphological” characters not found in the primary structure, enhancing the resolution and support of the phylogenetic hypotheses allowing solve relationships up to interfamiliar level. It is also important the information that secondary structures contributes for the correction of multiple alignments (Coleman 2003).

In this work, previously reported phylogenetic hypotheses of Caribbean octocorals were tested and complemented (Sanchez et al. 2003b, Wirshing et al. 2005) using ITS2 as molecular marker. To achieve that, ITS2 sequences of more than 60 samples from 23 species of octocorals were obtained and their secondary structure predicted. Sequences analyses were done using Maximum Likelihood and Bayesian Inference methods to obtained primary phylogenetic hypotheses. Maximum Parsimony analyses with combined primary and morphological characters were done for the combined phylogenetic hypotheses

Mate rials and Methods Sample collection, identification and DNA extraction. Octocoral samples were collect ed during May 2005 - Oct ober 2006 from six different locat ions bet ween Cart agena and Rosario Islands using SCUBA diving. Eight y samples of 25 species with emphasis in genus Eunicea, Gorgonia and Pseudopterogorgia were collect ed and preserved in absolute ethanol. Samples from other Caribbean locations were obtained from Biommar and Dr. J. A. Sanchez collect ions which were tagged and organized for this study (Refer to appendix A for Samples details). All samples collected were identified trough microscope comparison of sclerit es using the guide by Bayer (1961). Total DNA was extracted using a CTAB, proteinase K, Phenol – Clorophorm-IsoAmyl alcohol extraction method (Coffroth et al. 1992). DNA was resuspended and conserved in TE buffer at –70ºC. DNA quality was observed in agarose (1%) electrophoresis at 80V for 30 min. The extraction method was standardized, showing an efficiency of 90%. 27 DNA samples of Pseudopterogorgia bipinnata from previous st udies were also included in this work.

PCR A total of 177 DNA extractions samples of 29 species were chosen, including at least 6 samples per species that showed the best DNA extraction quality. Primers 5.8s 5` AGCAT GT CT GT CT GAGT GTT GG 3 ` an d 28 s 5 `GGGT AAT CTT GCCT GAT CT GAG3 ` designed by Aguilar and Sanchez (2007) were used for the IT S2 amplificat ion, condit ions for

27 PCR were as follows: an initial denaturizing step: 2 min at 94ºC, followed by 35 cycles: 30 sec. at 94ºC, 30 sec. at 56.8ºC and 1 min at 72ºC, final extension step: 2 min. at 72ºC. Using 1

unit of Taq polymerase (Invitrogen), 3.5 mM MgCl2, 0.2 mM DNTPs (Biorad Mix), 0.15 uM primers (each) and 4µL of DNA (dilution 1/50) in 20µL as final volume. The amplification was standardized with an efficiency of 95% in Pseudopterogorgia and Go rg on ia samples, and 65% in Eunicea Samples (Refer to Appendix B for PCR efficiency details).

Fig. 2.1 Predicted secondary structure of a sample of P. bipinnata, the structure corresponds to the conserved six-helicoidal ring- model. The characters used for the construction of the morphological matrix are shown: (N: Number, L: Letter) N stem, N` opposite side stem, Ni terminal loop, i` spacer, NL internal loop, N` L opposite side of the internal loop.

DGGE, Band extraction and DNA precipitation 114 PCR react ions of 23 species were separat ed in a gel cont aining 8% polyacrilamide, 1X TAE Buffer and linear urea-formamide denat uring gradient from 45% to 80%. Gels were pre-run at 60ºC and 90V for 30 min, followed by the electrophoresis at 60ºC and 90V for 13 h. The gels were stained wit h Et hidium Bromide during 15 min and visualized using a BIORAD chemidoc system. All the reactions were conducted without CG-clamp. Bands visualized in the DGGE gel were cut using sterilized micropipette tips in the BIORAD

28 chemidoc system and put in 0,5 mL tubes with 100µL of sterilized double distilled water, the tubes were placed in a shaker at room temperature for 24 hours at 150 rpm. Each band extract was the collected in a 0,5 mL tube and the DNA was precipitated with 300 µL of cold absolute Ethanol, tubes were placed at -20ºC for 24 hours and then centrifuged at 13000 rpm for 30 minut es, supernatant was discarded and pellet dried and resuspended in 15 µL st erilized double distilled water.

ITS2 sequences and prediction of ITS2 RNA secondary structures Amplifications of the DNA extracted from bands were conducted as described above except that DNA was used without dilution. Purification of PCR products for sequencing was performed by Exo-Sap method using 1u of Exonuclease, 0,2 u of Shrimp Alkaline Phosphatase and 2µL SAP buffer 10x per 20 µL in a 0,2 mL tube, reactions were held at 37ºC for 1 hour and at 80º for 15 minutes. Sequencing reactions were done with BigDye 3.1 system according to manufactures instructions (Applied Biosystems) and sequenced in a capillary electrophoresis automated sequencer (AB3100). Each sample was sequenced with forward and reverse primers, the consensus sequences were obtained by assembling the two complementary chromatograms in Sequencher 4.7 software. 42 ITS2 sequences of Octocorals were also obtained from GenBank for further analysis (refer to appendix A for samples details).

Secondary structures of all sequences obtained (including GenBank sequences) were reconstructed by comparison via Pairwise Alignment (Bioedit) with structures previously report ed in Octocorals (Aguilar and Sanchez 2007). T he sequences were then submitt ed wit h constrains and restrictions in MFOLD (Zuker, 2003) at a default temperature of 37ºC, the structure chosen was the one with greater negative free energy but with the ring model more similar to the reported. The obtained secondary structures were used to construct a matrix for cladistic analysis as described by Aguilar y Sanchez (2007). Number of nucleotides for each structure character were counted and arranged in discrete character states ranging from 0 to 9, were 0 is absence of the character and 1 the less nucleotide number (See Appendix C). A molecular morphometry phylogenetic and a combined morphometry–sequence hypothesis were obt ained by maximum parsimony using exhaust ive branch and bound algorithm in PAUP wit h a boot strap analysis of 1000 replicat es (Swofford 2002). For these analyses all sequences from other families different from Plexauridae and Gorgoniidae were excluded to reduce noise and possible long branch attraction on the trees as observed in primary sequences analyses (see fig. 2.3 and 2.4).

29 30 Fig. 2.2 (Previous page) Examples of ITS2 predicted secondary structures of different species. Is included ∆G and geographical location where the sample was taken are mentioned.

ITS2 sequence analysis All alignment s were performed using Clust alW, T -Cofee and Muscle wit h default parameters in the European Bioinformatics Institute web site (www.ebi.ac.uk), the best results compared with the secondary structures were obtained with Muscle. Alignments were eye corrected comparing with secondary structure characters. Maximum parsimony analyses were performed in PAUP with a Boot strap analysis of 1000 replicat es. Maximum Likelihood analyses were also made in PAUP with 100 replicates Bootstrap analysis according to the best model suggest ed in Modelt est. Bayesian inference of phylogeny was done using MrBayes according to the best model suggest ed in MrModelt est wit h 1000000 generat ions, sample frequency of 1000 and burning zone of 500. For Maximum Likelihood and Bayesian inference when Akaike Information Criterion (AIC) and Hierarchical Likelihood Ratio Test (hT LR) did not suggest the same model both models were used for the analysis and compared with Shimodaira-Hasegawa (SH) test in PAUP choosing the best result. The results obtained with the three methods were also compared with SH test to find the best phylogenetic hypot hesis.

Results ITS2 sequence and RNA predicted secondary Great variability in the length of the ITS2 copies were obtained, ranging form 154 bp t o 281 bp wit h an average of 236 bp (For more det ails see chapt er 1), unexpect edly bot h shorter and longer amplifications products were obtained from different DGGE bands of the same sample of Eunicea succinea (a complete reference of length of ITS2 of all samples and species used in this study including GenBank Sequences is included appendix C and D), demonstrating that ITS2 intragenomic differences could be remarkable; however intragenomic and intraspecific differences in ITS2 length were more often less abrupt in a range between 1 and 30 bp.

Predicted secondary structures followed the conserved six-helicoidal ring-model previously reported for octocorals (Aguilar & Sanchez 2006) (see fig. 2.1), but a great variability was observed in the length and complexity of each stem and spacer (see fig. 2.2). In general the stems II, III and VI are shorter than the stems IV and V, being the longest the stem V. Multiple internal loops are frequent in stems III, IV and V, with major number in stem V

31 32

Fig. 2.3 (Previous page) Bayesian inference of phylogeny of all samples including GenBank sequence (only sequences). Inference was done using MrBayes according to the model SYM+I+G (hTLR) suggested by MrModeltest with 1000000 generations, sample frequency of 1000 and burning zone of 500. Blue samples correspond to Gorgoniidae family, Black samples correspond to Plexauridae family and green were Alcyonacean octocorals as outgroups. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families. Posterior probability is also shown.

where up to six internal loops were observed. The spacers are more often short ranging from 1 to 4 bp, the spacer 1i showed a conserved sequence UG with little variations across the studied species, the spacer 4i is the longest with 4 to 12 bp with a conserved core sequenced AGUNCAGC observed in most of the samples studied, this conserved characteristics are probable main features recognized by processing machinery of rRNA. The morphometrics met hod applied to the predict ed secondary struct ure provided 43 parsimony-informat ive characters (see appendix E and F) according to PAUP.

Differences between copies of the same sample in most cases were discrete changes that do not affect the predicted secondary structure (see fig 1.2) while others dramatically change the ITS2 structure affecting the ∆G and even causing the disappearing of complete stem and/or loops (See fig 1.3a and 1.3b), the former changes were considered as allele variation while severe changes could be considered as pseudogenes; however only two sequences (G. flabellum and E. succinea) were finally considered as unquestionable pseudogenes because of the loss of characteristics that were present in all other secondary structures of the same sample and species. Other four sequences from Eunicea species were considered as possible pseudogenes for evident changes in the structure (including lower ∆G) and length of the sequence (for more details see chapter 1).

Alignm ents and selection of best phylogenetic analyses Alignment s of the sequences were performed using three different methods (T-Cofee, Muscle and ClustalW. For more details refer to Materials and Methods)), obtaining better results with Muscle. Alignments were corrected manually based on the conserved secondary structures, and also were eliminated some non-informative characters. Even with correction of the alignment, considerably number of INDELs was obtained as previously reported (i.e. Harris & Crandall 2000). The two pseudogenes found from E. succinea and G. flabellum (see chapter 1 for details) were not included in the alignments for their differences in length and structural characteristics, which could introduce high level of noise in the alignments.

33 34

Fig. 2.4 (Previous page) Maximum Likelihood hypothesis of all samples including GenBank sequence (only sequences). Inference was done using PAUP according to the model GTR+G+I (AIC) suggested by Modeltest, Majority rule (50%). Bootstrap 100. Blue samples correspond to Gorgoniidae family, Black samples correspond to Plexauridae family and green were Alcyonacean octocorals as outgroups. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families. Bootstrap is also shown.

From the edited Muscle alignments, phylogenetic analyses were performed using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. ML and BI where performed according to best model suggest ed by Modelt est and MrModeltest respectively, but the Akaike Information Criterion (AIC) and Hierarchical Likelihood Rat io Test (hT LR) never suggest ed t he same model, thus both models were run in every case. Trees obtained were compared with Shimodaira-Hasegawa (SH) test in PAUP to find if AIC and hTLR had significat ive differences and suggest ions for the best tree (dat a not shown). Trees obtained from the three methods were also compared with SH test. Always MP trees were considerably different with worse result compared to BI and ML trees (i.e. trees from MP: -ln L 8512.40 and ML: -ln L 8444.75 P: 0.00). MP and BI analyses were always considered similar with P always larger than 0.05 but in all cases ML always had a lower –ln L. According to all comparison performed, ML and BI trees were selected for phylogeny analyses (see figures 2.3 and 2.4 for models det ails).

Based on the morphometry matrix (see appendix E) MP analyses were performed (see methods for procedure details). Trees from these analyses showed low support and numerous polytomies even when all 43 characters were parsimony informative (Data not shown). The MP analysis that combined both primary and secondary characters showed better supports and less polytomies than morphometry trees. When the trees from combined MP analyses were compared with the primary sequence trees (ML and BI), it was clear that combined analyses (primary and morphological characters) offered bett er support , less polyt omies and more sense in the placement of some clusters, even when the combined hypotheses were obtained from MP analyses (see fig. 2.5 for more details) as reported previously (Aguilar & Sanchez 2007). However, in these comparisons it was not possible to apply the SH test due to differences in the sequences used.

Phylogenetic hypothesis All the phylogenetic hypotheses obtained showed the presence of conserved clusters with very strong supports (see fig. 2.3, 2.4, 2.5 and 2.6), the difference between hypotheses

35 consisted in the placement of clusters in the trees. The most remarkable result is the mix between Plexauridae and Gorgoniidae families in most clusters with very high support (bootstrap or posterior probability). In MP combined hypotheses it is suggest ed a Gorgoniidae predominant supercluster with some Eunicea species in it. This supercluster has significant support in the Gorgoniidae tree (See Fig. 2.6a). Other notable result is the placement in different clusters of species belonging to families different from Plexauridae and Gorgoniidae in bot h BI and ML hypot heses (see fig 2.3 and 2.4 in green). Both species of Clavuraiidae family included in this study (Clavularia koellikeri and Clavularia viridis) were placed at a considerable distance, C. koellikeri was placed with very good support with Calyptoptora japonica of family Primnoidae and E. clavigera while C. viridis was placed in a basal position without strong support (See fig. 2.3 and 2.4). All the four species of the family Nephteidae were placed in a single cluster with very strong support and Alcyonium digitatum of Alcyniidae family was placed with a cluster of Plexaura and Plexaurella.

Unexpectedly, none of the genuses Gorgonia, Pseudopterogorgia and Eunicea were placed in a single cluster, all were located in a wide distribution across the trees. However, some clusters contained most the species of each genus but mixed with other species. Pseudopterogorgia species were placed in three different clusters in all hypotheses; a main and exclusive P. bipinnata cluster with 90% of this species samples (described below), and the rest of P. bipinnata, P. acerosa and P. rigida were placed in two closely related mixed clusters wit h Gorgonian and Eunicea species (see fig. 2.3, 2.4, 2.5 and 2.6), probably these clusters are closer to Gorgoniidae, and according to MP combined hypothesis, these two joined with P. bipinnata clust er and Leptogorgia cluster could form a Gorgoniidae superclust er, but only strong support for this was obtained in Gorgoniidae tree (see fig. 2.6a). Go rg on ia species are widespread in all trees, but most of the samples were placed in the two closely related mixed clusters, other sample was placed in the Muriceopsis – Pterogorgia cluster while the last one was placed in a cluster with Plexaurella, Eunicea and Eunicella (see fig. 2.3, 2.4 and 2.5).

Most of the Eunicea samples were placed in two main clusters, the first one is basal according to BI and ML analyses grouped together with Sclerobelemnon theseus with very high support (see fig. 2.3, 2.4, 2.5 and 2.6b), t his cluster cont ained all possible pseudogenes and ot her candidat es to pseudogenes (see det ails in chapt er 1). T he other Eunicea highly supported cluster is composed by eight samples of E. fusca, E. laciniata, E. tourneforti, E. succinea and E. flexuosa and was placed close to the mixed cluster of Muriceopsis – Pterogorgia. The rest of Eunicea samples were placed in the two mixed cluster with conserved secondary structures.

36 37

Fig. 2.5 (Previous page) Maximum Parsimony Combined hypothesis of primary and morphological characters. Inference was done using PAUP, Majority rule (50%). Bootstrap 1000. 279 parsimony-informative characters from 370. Blue samples correspond to Gorgoniidae family, Black samples correspond to Plexauridae family. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families, conserved secondary structure is also shown. Bootstrap results are shown.

A group very well defined is a P. bipinnata cluster which included t he 90% of P. bipinnata sequences, all from Belize and Panama; it is important that in these sequences were included t hree different morphot ypes (deep, shallow and int ermediat e) previously reported (Sanchez et al. 2007). This cluster is very homogeneous in its secondary structures (see chapt er 1), and the organizat ion of this clust er has no evident relat ion with the morphot ypes or wit h the geographical locat ion of the samples (see fig. 2.3, 2.4, 2.5 and 2.6a). Ot her P. bipinnata samples were placed with a mixed group of the two conserved alleles of Plexauridae and Gorgoniidae species, all P. bipinnata samples that were placed outside the cluster were collected from different locations.

Leptogorgia (=Lophogorgia) and Pacifigorgia species were placed in a highly supported cluster as previously reported (Aguilar & Sanchez 2007) (See fig. 2.3 and 2.4). Other strong group is the Plexaura-Pseudoplexaura cluster where A. digitatum is placed but no Plexaurella nutants (see fig. 2.3 and 2.4). The cluster composed by Muriceopsis species and Pterogorgia citrina was very strong and was observed in all trees as previously reported (Wirshing et al. 2005), additionally Pinnigorgia species were added to this cluster according to BI (see fig. 2.3).

Discussion ITS2 secondary structure and alignments The use of morphometry of ITS2 secondary structures as independents morphological characters improved the support of the branches and reduce the polytomies as previously reported (i.e. Chen et al. 2004, Aguilar & Sanchez 2007, Grajales et al. 2007) thus the consistency and resolution of the trees are enhanced allowing the use of ITS2 to find relationships of higher groups, comparing to the previously reported saturation of ITS2 at intrageneric level (Aguilar & Sanchez 2007). But, according to the results obtained, the limit for the usefulness of ITS2 is Interfamiliar analyses, due that not strong basal supports were obtained (as bootstrap and posterior probabilities) (see fig. 2.3, 2.4 and 2.5). However, when fewer samples were used of only Plexauridae and Gorgoniidae families, better support for

38 basal ramificat ions were obtained (see fig 2.6), probably due to noise reduct ion and less Long Branch attraction effect (Bergsten 2005).

It was demonstrated the usefulness of secondary structures for uses as detection of pseudogenes (which probably could increase the noise of alignments) and correction of alignments according to conserved structures (Marquez et al. 2003). It was also clear that all met hods t o perform mult iple alignment s produce different result s, and as previously report ed (Robert 2004) Muscle produced the best alignments according to the conserved features of the predicted secondary structures, in the other side ClustalW presented the alignments with less resemblance with the conserved features. Interesting this program is still the most used algorithm for all purposes even when its deficiencies have been widely reported (Robert 2004).

When multiple sequences per sample and/or species are used in a phylogenetic analysis, the noise could be increased. This was seen when different sequences of the same sample were placed in different clusters (see fig. 2.3 and 2.4). Probably this is due to the presence of pseudogenes that are not very different from the functional alleles and are not easily recognized. Thus, it is highly recommended from upcoming works to make strict searches for pseudogenes and reject them for phylogenetic analyses for the reduction of alignment noise.

Phylogenetic analyses The use of ITS2 as independent molecular marker confirmed the results obtained by Sanchez et al. (2003b) and Wirshing (2005) where families Plexauridae and Gorgoniidae where described as polyphyletic groups, seen as mixed cluster across the obtained trees. This indicates the urgent need for a revision of the placement of the different genuses in the families. It is also recommended to realize further studies in the family Clavulariidae because both species studied were placed at considerable distance even when they belong to same genus (See fig. 2.3 and 2.4). Cont rary all the four family Nepht eidae species used in this study were placed in a single cluster with very strong support, which is clear indication of a monophyletic family. The presence of different families within the Plexauridae and Gorgoniidae families seen in fig. 2.3 and 2.4 probably reflects the saturation of ITS2 sequence at the interfamiliar level as previously reported (Aguilar & Sanchez 2007), and also Long branch attraction phenomena, which were reduce using morphological characters from secondary structures and excluding divergent sequences as discussed above.

39 A very important cluster of P. bipinnata with very strong support included most of this species samples, but the most remarkable result is that within this group samples of three

40

41 Fig. 2.6 (Previous page) Maximum Parsimony Combined hypothesis of primary and morphological characters for Gorgoniidae (a.) and Plexauridae (b) families. Inference was done using PAUP, Majority rule (50%). Bootstrap 1000. Blue samples correspond to Gorgoniidae family and Black samples correspond to Plexauridae family. Yellow arrows indicate the probable pseudogenes found. Blue lines indicate the clusters. Clusters a and b correspond to two conserved ITS2 alleles between Plexauridae and Gorgoniidae families. Bootstrap results are shown. a. Gorgoniidae tree. 225 parsimony-informative characters from 337. b. Plexauridae tree. 268 parsimony-informative characters from 374

different morphotypes found in Belize and Panama at different depths were placed (see fig. 2.3, 2.4, 2.5 and 2.5a). These morphotypes were reported by Sanchez et al. (2007) concluding that the morphological variability was due to phenotypic plasticity. My results supports Sanchez at al. (2007) hypothesis due to the complete homogeneity presented in the ITS2 of different P. bipinnata morphotypes; this was seen as conserved ITS2 secondary structures reflected in a compact cluster with very high support that includes all P. bipinnata samples. This is also supported by the fact that P. bipinnata samples from other locations (Bahamas and Colombia) with standard morphotypes presented a great ITS2 variability, suggest ing that in this species changes in morphology depend in environmental conditions as depth. The diversity of IT S2 sequences in this species from dist ant locat ion could be explained by incomplete lineage sorting phenomena of ITS2 and/or the reproductive isolation of populations by geographical and physical barriers as sea currents, islands and rivers mouths. However, the results obtained in this study are not enough conclusive to confirm any of these hypotheses, thus phylogeographic studies are needed to gather information to solve this questions.

Ot her smaller but no less import ant group is the Muriceopsis and Pt erogorgia cluster, which also includes in the BI and ML hypotheses the Pinnigorgia genus (see fig. 2.3, 2.4 and 2.5), this clust er had been report ed previously using mult iple mit ochondrial loci (msh1, ND2 and ND6) (Sanchez et al. 2003b, Wirshing et al. 2005). This well supported group is composed by species that traditionally belong to Plexauridae and Gorgoniidae families, giving support to a needed reorganization of theses families. In the same papers Sanchez (2003) and Wirshing (2005) also described the genus Plexaurella in the Gorgoniidae family which traditionally was placed close to genuses Plexaura and Pseudoplexaura in the Plexauridae family, in this work was found t hat Plexaura and Pseudoplexaura were placed in a strong cluster with E. mammosa (BI and ML) but Plexaurella was placed in mixed cluster with other Eunicea, Eunicella, Tobagogorgia and close with the Muriceopsis – Pterogorgia cluster. Thus, without any doubt Plexaurella is not a closely related genus to the Plexaura-Pseudoplexaura group. Other group found with very strong support in all analyses was the Leptogorgia (=Lophogorgia)-Pacifigorgia clust er. Result s support ed this group as monophylet ic, but it s

42 placement is not well defined, the most likely hypothesis (MP combined se fig 2.5 and 2.6a) is that this group is placed at basal position with the P. bipinnata cluster.

Eunicea species were widespread on the trees; however a well defined group of Eunicea ITS2 copies were placed together as basal group (See fig. 2.3 and 2.4), most of these sequences have been described as possible pseudogenes or candidat es to pseudogenes, thus is likely that this group of sequences are evolutive remnants from a common ancestor (see chapter 1 for details), the basal position and the placement of these sequences with S. theseus in ML an BI hypotheses support this idea. But, the big distance seen in BI tree and no apparent similitude between secondary structures within this cluster, could also suggest a long branch attraction phenomena. Other well supported Eunicea clust er that added addit ional evidence of the polyphyletic relations included samples of E. fusca, E. laciniata, E. tourneforti, E. succinea and E. flexuosa joined with Filigorgia africana (Gorgoniidae species), which is placed in BI and ML hypotheses close to Dendronephthya species but far from other Eunicea species. Other Eunicea species were placed in mixed clusters; all these results generated doubt s about the monophylet ic relat ionship of the genus, but also could be explained as in the P. bipinnata case, by incomplete lineage sorting and/or isolation by physical barriers.

The most unexpected result is the presence of two conserved alleles between the Plexauridae and Gorgoniidae families (See chapter 1 and fig. 2.3, 2.4 and 2.5), as expected, two clusters were obtained each included all samples that presented the conserved ITS2 characteristics. These clusters according to MP and ML analyses are closely related, and probably are closer to Gorgoniidae as members of the Gorgoiidae supercluster seen in combined analyses (fig. 2.5 and 2.6a). Explanations for the presence of these clusters were discussed in chapter 1, but with the observed results these clusters are real and not the result of artifacts or other problems. Thus there are multiple indications of the polyphyletic relation between these families.

The results obtained in this work are a clear indication of the difficulties to find the relat ionships of the octocorals, and reflect s the urgent need for the reorganizat ion of the families Plexauridae and Gorgoniidae. To find better phylogenetic hypotheses it is recommended to use multiple samples from different locations per species and also the combination of morphological and molecular characters. As seen in this study a lack of basal support of the ITS2 based phylogenetic hypotheses was obvious and thus better resolution could be obtained using less variable molecular markers as msh1 and ND6 together with the IT S2. It is also suggest ed to perform a phylogeographical work with only a couple of species

43 but including more samples from different locat ions to understand the effect of physical barriers in the gene flow of Caribbean octocorals and to confirm or deny phenomena as incomplete lineage sorting.

Acknowledgements This st udy was part ially funded by COLCIENCIAS (No. 120409-16825) and the Facultad de Ciencias, Department of Biological Sciences, Universidad de los Andes (funding to J.A. Sánchez). We are grateful with BIOMMAR divers and colleagues for their assistant during field surveys and laborat ory assays.

References Aguilar, C., Sánchez, J.A. (2007) Phylogenetic hypothesis of gorgoniid octocorals according to ITS2 and their predicted RNA secondary structures. Mol. Phylogenet. Evol. 43:774-786.

Bayer, F.M. (1961) The shallow water Octocorallia of the West Indian region. Studies of the Fauna of Curaçao 12, 1-373. Bayer, F.M. (1981) Status of knowledge of octocorals of world seas. Seminarios de Biología Marinha. Academia Brasileria de Ciencias, rio de Janeiro. 3-11. Bergsten, J. (2005) A review of Long-Branch Attraction. Cladistics 21 (2005) 163-193 Berntson, E.A., Bayer, F.M., McArthur, A.G., France, S.C. (2001) Phylogenetic relationships within the Octocorallia (Cnidaria: Anthozoa) based on nuclear 18S rRNA sequences. Marine Biology 138, 235-246. Chen, C. A., C. C. Chang, N. V. Wei, C. H. Chen, Y. T. Lein, H. E. Lin, C. F. Dai, C. C. Wallace (2004) Secondary structure and phylogenetic utility of the ribosomal internal transcribed spacer 2 (ITS2) in Scleractinean Corals. Zoological Studies 43, 759-771. Coffroth, M.A., Lasker, H.R., Diamond, M.E., Bruenn, J.A., Bermingham. E. (1992) DNA fingerprint s of a gorgonian coral: A method for det ect ing clonal struct ure in a veget at ive species. Marine Biology 114, 317-325. Coleman, A.W. (2003) IS2 is a double-edged t ool for eukaryot e evolutionary comparisons. Trends in Genetics Vol 19 Coté, C.A., Peculis, B.A. (2001) Role of the ITS2-proximal stem and evidence for indirect recognition of processing sites in pre-rRNA processing in yeast. Nucleic Acids Research 29, 2106-2116 Dorado, D. (2006) Uso del Espaciador Trascrito Interno 2 (ADNr) como marcador de especies de octocorales. Universidad de los Andes, Biology Thesis 2006. France, S.C., Rosel, P.E., Agenbroad, J.E., Mullineaux, L.S., Kocher, T.D. (1996) DNA sequence variat ion of mitochondrial large-subunit rRNA provides support for a two-subclass

44 organization of the Anthozoa (Cnidaria). Molecular Marine Biology & Biotechnology 5, 15- 28. Grasshoff, M. (1999) The shallow water gorgonians of New Caledonia and adjacent islands (Coelenterata: Octocorallia). Senkenbergiana Biologica 78, 1-121. Grajales, A., Aguilar, C., Sanchez, J. (2007) Phylogenetic reconstruction using secondary structures of Internal Transcribed Spacer 2 (ITS2, rDNA): finding the molecµLar and morphological gap in Caribbean gorgonian corals. BMC Evolutionary Biology, 7:90 Harris, D.J., Crandall, K.A. (2000) Intragenomic variation within ITS1 and ITS2 of freshwater crayfishes (Decapoda: Cambaridae): Implications for phylogenetic and microsatellite st udies. Molecular Biology & Evolution 17, 284-291. McFadden, C.S., Tullis, I.D., Hutchinson, M. B., Winner, K., Sohm, J. A. (2004) Variation in coding (NADH dehydrogenase subunit s 2, 3 and 6) and non-coding int ergenic spacer regions of the mitochondrial genome in Octocorallia (Cnidaria: Anthozoa). Marine Biotechnology 6, 516-526. Mcfadden C. S., France, S. C., Sánchez, J.A. & Alderslade, P. (2006) A Molecular Phylogenetic Analysis of the Octocorallia (Cnidaria: Anthozoa) Based on Mitochondrial Prot ein-coding Sequences. Molecular Phylogenetics & Evolution 41, 3, 513-527. Robert, C.E. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acid Research Vol. 32 No 5 Sanchez, J.A., Lasker, H.R., Taylor, D.J. (2003a) Phylogenetic analyses among octocorals (Cnidaria): mitochondrial and nuclear DNA sequences (lsu-rRNA, 16s and ssu_rRNA, 18s) support t wo convergent clades of branching gorgonians. Molecular Phylogenetics and Evolution 29 31-42. Sanchez, J.A., McFadden, C.S., France, S.C. & Lasker, H.R. (2003b) Molecular phylogenetic analyses of shallow-water Caribbean Octocorals. Marine Biology 142, 975-987. Sanchez, J.A., Aguilar, C., Dorado, D., Manrique, N. (2007) Phenotypic plasticity and morphological integrat ion in a marine modular invertebrate. BMC Evolutionary Biology 7:122. Swofford, D. (2002) PAUP*, phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sunderland: SinauerAssociates. Wirshing, H.H., Messing, C.G., Douady, C.J., Reed, J., Stanhope, M.J., Shivji, M.S. (2005) Molecular evidence for multiple lineages in the gorgonian family Plexauridae (Anthozoa: Octocorallia). Marine Biology 147, 497-508. Zuker, M., (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406-3415.

45 APPENDIXES

Appendix A PCR efficiency: Efficiency of the PCR on the main genuses from the study. Eunicea species details are showed. Information included: Species or Genus, N (Number of samples tried), Amplified (Number of samples amplified) and % percentage of successfully amplified samples.

Sp./Genus N Amplified % Eunicea E. asperula 8 4 50 E. calyculata 8 2 25 E. clavigera 8 4 50 E. colombiana 10 5 50 E. flexuosa 8 8 100 E. fusca 8 4 50 E. Knighti 6 0 0 E. laciniata 9 3 33,3 E. laxispica 5 4 80 E. mammosa 6 6 100 E. pallida 7 6 85,7 E. palmeri 4 3 75 E. sp1 5 2 40 E. sp2 4 0 0 E. succinea 8 7 87,5 E. sp. 2 8 0 0 E. tourneforti 6 0 0 Eunicea 118 58 49,2 Gorgonia 17 17 100 Pseudopterogorgia 42 39 92,9 Total 177 114 64,4

Appendix B Sample details: Samples success fully amplified and run in DGGE. Information included: ID, Specie, Deep where was taken the sample (When available), Place and Country and Source of the sample.

ID Sp. Depth (ft) Place, Country Source B1 Gorgonia fl abellum Swan island Biommar B2 Gorgonia fl abellum Swan island Biommar 50421 Gorgonia mariae 6 Netherlands Antilles, St. Eustatius Island Smithsonian 51027 Gorgonia mariae 3 British Virgin Islands , A negada Isl and, P omat o P oint Smiths oni an 51351 Gorgonia mariae 3 Puert o Rico, Boqueron, Santurce Smiths oni an B108 Gorgonia mariae Swan island Biommar B117 Gorgonia mariae Boca del Drago Biommar B118 Gorgonia mariae Boca del Drago Biommar B127 Gorgonia mariae Boca del Drago Biommar A679 Gorgonia vent alina 45 Montañita, Cartagena Collected B377 Gorgonia vent alina 40 Tortugas, Cart agena Collected B458 Gorgonia vent alina 50 Salmedina, Cart agena Collected N213 Gorgonia vent alina San Bernardo - Llantas Biommar Oct 15 Gorgonia vent alina 40 Playita - Cart agena Collected Oct 50 Gorgonia vent alina 40 Burbuj as - Cartagena Collected

46 54 Gorgonia vent alina Akumal - Mexico Biommar B164 Gorgonia vent alina Punt a Vi eja Biommar N140 Pseudopterogorgia ac erosa 15 San Bernardo - Llantas Biommar N141 Pseudopterogorgia ac erosa 15 San Bernardo - Llantas Biommar P71 Pseudopterogorgia ac erosa Bahamas Biommar B124 Pseudopterogorgia ac erosa Boca del Drago Biommar A715* Pseudopterogorgia americana 45 Montañita, Cartagena Collected A471 Pseudopterogorgia bi pinnata 60 Salmedina, Cart agena Collected P32 Pseudopterogorgia bi pinnata Bahamas Biommar 96216 Pseudopterogorgia bi pinnata 8 Bahamas, Eleut hera Island Biommar Pbip25 Pseudopterogorgia bi pinnata Int Belize Biommar Pbip13 Pseudopterogorgia bi pinnata Sha Belize Biommar Pbip15 Pseudopterogorgia bi pinnata Sha Belize Biommar Pbip6 Pseudopterogorgia bi pinnata Sha Belize Biommar Pbip53 Pseudopterogorgia bi pinnata Belize Biommar Pbip14 Pseudopterogorgia bi pinnata Sha Belize Biommar Pbip1 Pseudopterogorgia bi pinnata Belize Biommar Pbip2 Pseudopterogorgia bi pinnata Belize Biommar Pbip23 Pseudopterogorgia bi pinnata Deep Belize Biommar Pbip24 Pseudopterogorgia bi pinnata Deep Belize Biommar Pbip22 Pseudopterogorgia bi pinnata Deep Belize Biommar Pbip27 Pseudopterogorgia bi pinnata Deep Belize Biommar Pbip7 Pseudopterogorgia bi pinnata Sha Belize Biommar Pbip3 Pseudopterogorgia bi pinnata Belize Biommar Pbip20 Pseudopterogorgia bi pinnata Deep Belize Biommar Bip50B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip47B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip54B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip44B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip71 Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip72 Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip41B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip46B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip40B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip45B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar Bip49B Pseudopterogorgia bi pinnata Cristobal, P anama Biommar 100594 Pseudopterogorgia bl anquillensis Navassa Island, NW Poi nt Biommar P2 Pseudopterogorgia elisabet hae Bahamas Biommar 80951 Pseudopterogorgia oppositipinna 12 Australia, Northern Territor y, S andy Island No. 2 Smiths oni an A511* Pseudopterogorgia rigida 40 Burbuj as, Cart agena Collected A538* Pseudopterogorgia rigida 65 Burbuj as, Cart agena Collected A475 Eunicea asper ula 60 Salmedina, Cart agena Collected T2 Eunicea asper ula 30 Trinidad y T obago Biommar T88 Eunicea asper ura 36 Trinidad y T obago Biommar Oct 37 Eunicea asper ura gruesa 40 Burbuj as - Cartagena Biommar 8 Eunicea c alyc ulat a 60 Trinidad y T obago Biommar CAT22 Eunicea c alyc ulat a Akumal - Mexico Biommar A129 Eunicea clavigera 35 Salmedina, Cart agena Collected 28 Eunicea clavigera Akumal - Mexico Biommar 50076 Eunicea clavigera Bermuda, North Roc ks Smiths oni an T73 Eunicea clavigera 60 Trinidad y T obago Biommar A472 Eunicea c olombiana 60 Salmedina, Cart agena Collected A692 Eunicea c olombiana 45 Montañita, Cartagena Collected 107 Eunicea c olombiana Akumal - Mexico Biommar Oct 17 Eunicea c olombiana grues a 40 Playita - Cart agena Collected Oct 45 Eunicea c olombiana grues a 40 Burbuj as - Cartagena Collected A223 Eunicea fl exuos a 40 Isla Tesor o, Cart agena Collected A545* Eunicea fl exuos a 65 Burbuj as, Cart agena Collected A716 Eunicea fl exuos a 45 Montañita, Cartagena Collected Oct 41 Eunicea fl exuos a 40 Burbuj as - Cartagena Collected Oct 51 Eunicea fl exuos a 40 Burbuj as - Cartagena Collected P9 Eunicea fl exuos a Bahamas Biommar P75 Eunicea fl exuos a Bahamas Biommar M19B Eunicea fl exuos a San Sal vador Biommar A480 Eunicea f usca 60 Salmedina, Cart agena Collected A689 Eunicea f usca 45 Montañita, Cartagena Collected Oct 40 Eunicea f usca 40 Burbuj as - Cartagena Collected B142 Eunicea fusca Cayo Aqua NW Biommar A482 Eunicea laci niata 60 Salmedina, Cart agena Collected A706 Eunicea laci niata 45 Montañita, Cartagena Collected

47 Oct 42 Eunicea laci niata 40 Burbuj as - Cartagena Collected A606 Eunicea laxis pica 65 Imel da, C artagena Collected P62 Eunicea laxis pica Bahamas Collected 51557 Eunicea laxis pica 45 Virgin Islands of t he U nited St at es, S t. John Island Smiths oni an 51739 Eunicea laxis pica Virgin Islands of t he U nited St at es, S t. John Island Smiths oni an Oct 21 Eunicea mammosa 40 Burbuj as - Cartagena Collected P61 Eunicea mammosa Bahamas Biommar CAT18 Eunicea mammosa Akumal - Mexico Biommar CAT21 Eunicea mammosa Akumal - Mexico Biommar B119 Eunicea mammosa Boca del Drago Biommar B141 Eunicea mammosa Cayo Aqua NW Biommar Oct 26 Eunicea pallida 40 Burbuj as - Cartagena Collected Oct 27 Eunicea pallida 40 Burbuj as - Cartagena Collected MI25 Eunicea pallida Tesoro Biommar MI141 Eunicea pallida Isla Fuert e Biommar B178 Eunicea pallida Crawl Cay Biommar T68 Eunicea pallida 60 Trinidad y T obago Biommar 82140 Eunicea palmeri 5 Florida Keys , Looe K ey Smiths oni an 82140 Eunicea palmeri 5 Florida Keys , Looe K ey Smiths oni an 82140 Eunicea palmeri 5 Florida Keys , Looe K ey Smiths oni an Oct 60 Eunicea s p1 20 Salmedina - Cartagena Biommar PA98 Eunicea s p1 Panama Biommar A252 Eunicea s uccinea 40 Isla Tesor o, Cart agena Collected Oct 28 Eunicea s uccinea 40 Burbuj as - Cartagena Collected Oct 32 Eunicea s uccinea 40 Burbuj as - Cartagena Collected Oct 77 Eunicea s uccinea 20 Salmedina - Cartagena Collected #36 Eunicea s uccinea Bahamas Biommar 51555 Eunicea s uccinea 6 Virgin Islands of t he U nited St at es, S t. John Island Smiths oni an 80836 Eunicea s uccinea 9 St. Vi ncent and the Grenadines, Tobago Cays Smiths oni an

Appendix C Details of sequences used for the prediction of the ITS2 RNA secondary structure: Specie, ID (Laboratory identification or GenBank ID), Family, Suborder, Order, ITS2 length, Place and Country.

Spp. ID Band Family SubOrder ITS2 Size Country Alcyonium digitatum EF090736. 1 Alcyoniidae 253 U Clavularia koellikeri AB055932.1 ClavµLariidae 272 Japan Clavul aria viridis AB055931.1 ClavµLariidae 254 Japan Dendronepht hya c ast anea AF320101. 1 Nephtheidae 250 U Dendronepht hya gigant ea AF320104. 1 Nephtheidae 250 U Dendronepht hya s pinif era AF320106. 1 Nephtheidae 251 U Dendronepht hya s uensoni AF320096. 1 Nephtheidae 241 U Eunicella cavolinii AY827535.1 Gorgoniidae Holaxonia 217 France Eunicella singµLaris AY827540.1 Gorgoniidae Holaxonia 219 France Filigorgia africana AY587532.1 Gorgoniidae Holaxonia 229 Angola Gorgonia fl abellum AY587521.1 Gorgoniidae Holaxonia 241 Bahamas Gorgonia fl abellum B1 B2 Gorgoniidae Holaxonia 201 Panama Gorgonia fl abellum B1 B3 Gorgoniidae Holaxonia 247 Panama Gorgonia mariae AY587523.1 Gorgoniidae Holaxonia 245 Puert o Rico Gorgonia mariae B108 B1 Gorgoniidae Holaxonia 207 Panama Gorgonia mariae B108 B2 Gorgoniidae Holaxonia 222 Panama Gorgonia mariae B127 B2 Gorgoniidae Holaxonia 240 Panama Gorgonia vent alina AY587522.1 Gorgoniidae Holaxonia 241 Puert o Rico Gorgonia vent alina A679 Gorgoniidae Holaxonia 243 Colombia Gorgonia vent alina N213 B6 Gorgoniidae Holaxonia 233 Colombia Leptogorgia set acea AY587525.1 Gorgoniidae Holaxonia 233 USA Leptogorgia vi olacea AY587527.1 Gorgoniidae Holaxonia 235 Trinidad y Tobago Leptogorgia virgµLat a AY587526.1 Gorgoniidae Holaxonia 233 USA Lophogorgia euryale AY587530.1 Gorgoniidae Holaxonia 231 Panama Lophogorgia mi niata AY587528.1 Gorgoniidae Holaxonia 228 Trinidad y Tobago Pacifigorgia stenobroc his AY587531.1 Gorgoniidae Holaxonia 232 Easter n P acific Ocean Pinnigorgia flava AY587535.1 Gorgoniidae Holaxonia 233 Palau Pinnigorgia plat ys oma AY587536.1 Gorgoniidae Holaxonia 236 Australia Pseudopterogorgia ac erosa P71 Gorgoniidae Holaxonia 217 Bahamas Pseudopterogorgia bi pinnata Pb13 B1 Gorgoniidae Holaxonia 219 Belize Pseudopterogorgia bi pinnata P32 88 Gorgoniidae Holaxonia 249 Bahamas Pseudopterogorgia bipinnata A471 Gorgoniidae Holaxonia 246 Cartagena

48 Pseudopterogorgia bi pinnata Pb40B Gorgoniidae Holaxonia 220 Panama Pseudopterogorgia bi pinnata Pb41B Gorgoniidae Holaxonia 220 Panama Pseudopterogorgia bi pinnata Pb22 B3 Gorgoniidae Holaxonia 212 Belize Pseudopterogorgia bi pinnata Pb7 B1 Gorgoniidae Holaxonia 213 Belize Pseudopterogorgia bi pinnata Pb23 B2 Gorgoniidae Holaxonia 219 Belize Pseudopterogorgia bi pinnata Pb20 B1 Gorgoniidae Holaxonia 218 Belize Pseudopterogorgia bi pinnata Pb47 B1 Gorgoniidae Holaxonia 217 Panama Pseudopterogorgia bi pinnata Pb6 B3 Gorgoniidae Holaxonia 224 Belize Pseudopterogorgia bi pinnata Pb20 B3 Gorgoniidae Holaxonia 217 Belize Pseudopterogorgia bi pinnata Pb44 B2 Gorgoniidae Holaxonia 218 Panama Pseudopterogorgia bi pinnata PBIP 25 P14 Gorgoniidae Holaxonia 217 Belize Pseudopterogorgia bi pinnata PBIP 1 Gorgoniidae Holaxonia 220 Belize Pseudopterogorgia bipinnata Pbip53 Gorgoniidae Holaxonia 220 Belize Pseudopterogorgia bi pinnata AY587524.1 Gorgoniidae Holaxonia 233 Bahamas Pseudopterogorgia bi pinnata EU043123.1 Gorgoniidae Holaxonia 218 Belize Pseudopterogorgia rigida A511 Gorgoniidae Holaxonia 233 Cartagena Pseudopterogorgia rigida A538 Gorgoniidae Holaxonia 234 Cartagena Pterogorgia citrina EF065679. 1 Gorgoniidae Holaxonia 211 U Tobagorgia hardyi AY587534.1 Gorgoniidae Holaxonia 224 Trinidad y Tobago Alas kagorgia aleutiana EF090733. 1 Plexauridae Holaxonia 229 U Eunicea clavigera A129 Plexauridae Holaxonia 241 Cartagena Eunicea fl exuos a A716 Sa10B2 Plexauridae Holaxonia 254 Cartagena Eunicea fl exuos a P75 Sa14Bu Plexauridae Holaxonia 251 Bahamas Eunicea fl exuos a EF490984. 1 Plexauridae Holaxonia 216 U Eunicea f usca EF490983. 1 Plexauridae Holaxonia 253 U Eunicea laci niata A706 E2B 1 Plexauridae Holaxonia 232 Cartagena Eunicea laci niata EF090732. 1 Plexauridae Holaxonia 266 U Eunicea laxis pica 51557 E3B 3 Plexauridae Holaxonia 233 EUA Eunicea laxis pica EF490980. 1 Plexauridae Holaxonia 225 U Eunicea mammosa P61 E6B 4 Plexauridae Holaxonia 211 Bahamas Eunicea mammosa EF490979. 1 Plexauridae Holaxonia 241 U Eunicea pallida B178 E5B 1 Plexauridae Holaxonia 234 Panama Eunicea pallida B178 E5B 2 Plexauridae Holaxonia 257 Panama Eunicea pallida B178 E5B 3 Plexauridae Holaxonia 255 Panama Eunicea pallida B178 E5B 5 Plexauridae Holaxonia 235 Panama Eunicea pallida MI141 E6B 3 Plexauridae Holaxonia 243 Colombia Eunicea pallida MI25 E7B 1 Plexauridae Holaxonia 241 Cartagena Eunicea pallida MI25 E7B 2 Plexauridae Holaxonia 244 Cartagena Eunicea pallida Oct-26 E9B 2 Plexauridae Holaxonia 212 Cartagena Eunicea palmeri 82140C F3B1 Plexauridae Holaxonia 233 EUA Eunicea pallida EF490977. 1 Plexauridae Holaxonia 222 U Eunicea s p1 PA98 F2B1 Plexauridae Holaxonia 240 Panama Eunicea s uccinea Oct-28 F5B3 Plexauridae Holaxonia 228 Cartagena Eunicea s uccinea Oct-32 F6B2 Plexauridae Holaxonia 270 Cartagena Eunicea s uccinea Oct-32 F6B5 Plexauridae Holaxonia 208 Cartagena Eunicea s uccinea Oct-32 F6B6 Plexauridae Holaxonia 154 Cartagena Eunicea s uccinea 51555 F9B1 Plexauridae Holaxonia 233 EUA Eunicea s uccinea 80836 F10B 2 Plexauridae Holaxonia 281 Tobago Eunicea t ourneforti EF490982. 1 Plexauridae Holaxonia 245 U Muriceopsis bayeri AY587538.1 Plexauridae Holaxonia 258 Panama Muriceopsis flavi da AY587537.1 Plexauridae Holaxonia 247 Bahamas Plexaur a homomalla EF490974. 1 Plexauridae Holaxonia 210 U Plexaur a kuna EF090737. 1 Plexauridae Holaxonia 236 U Plexaur a kuna EF490975. 1 Plexauridae Holaxonia 208 U Plexaur ella nutans EF065680. 1 Plexauridae Holaxonia 217 U Pseudoplexaura crucis EF490973. 1 Plexauridae Holaxonia 216 U Calyptrophora j aponica EF090735. 1 Primnoidae Holaxonia 265 U Sclerobelemnon t hes eus EF090739. 1 Kophobelemnidae Sessiliflor ae 257 U 232,6

49 Appendix D ITS2 Length details of sequences used for the prediction of ITS2 secondary structure: A. length (Average ITS2 length in the correspond group) and N (number of samples in the group).

Order SubOrder Family Genus Speci e A. Lenght N Alcyonacea 253 7 Alcyonii dae Alcyonium Alcyonium digitatum 253 1 Clav ulariidae Clavularia 263 2 Clavularia koellikeri 272 1 Clavularia viridis 254 1 Nephteidae Dendronephthya 248 4 Dendronephthya castanea 250 1 Dendronephthya gigantea 250 1 Dendronephthya spinifera 251 1 Dendronephthya suensoni 241 1 Gorgonacea Holaxonia 232 80 Gorgoniidae 226 43 Eunicella 218 2 Eunicella cavolinii 217 1 Eunicella singµLaris 219 1 Filigorgia 229 1 Gorgonia 232 10 Gorgonia flabellum 232 3 Gorgonia mariae 229 4 Gorgonia ventalina 242 3 Leptogorgia 234 3 Leptogorgia setacea 233 1 Leptogorgia violacea 235 1 Leptogorgia virgµLata 233 1 Lophogorgia 230 2 Lophogorgia euryale 231 1 Lophogorgia miniata 228 1 Pacifigorgia Pacifigorgia stenobrochis 232 1 Pinnigorgia 235 2 Pinnigorgia flava 233 1 Pinnigorgia platysoma 236 1 Pseudopterogorgia 223 22 Pseudopterogorgia acerosa 217 1 Pseudopterogorgia bipinnata 222 19 Pseudopterogorgia rigida 234 2 Pterogorgia Pterogorgia citrina 211 1 Tobagogorgia Tobagorgia hardyi 224 1 Plexauridae 235 36 Alaskagorgia Alaskagorgia aleutiana 229 1 Eunicea 236 29 Eunicea clavigera 241 1 Eunicea fusca 253 1 Eunicea laciniata 249 2 Eunicea laxispica 229 2 Eunicea mammosa 226 2 Eunicea pallida 238 10 Eunicea palmeri 233 1 Eunicea sp1 240 1 Eunicea succinea 229 6 Eunicea tourneforti 245 1 Musiceopsis 253 2 Muriceopsis bayeri 258 1 Muriceopsis flavida 247 1 Plexaura 218 3 Plexaura homomalla 210 1 Plexaura kuna 222 2 Plexaurella Plexaurella nutans 217 1 Pseudoplexaura Pseudoplexaura crucis 216 1 Primnoidae Calyptrophora Calyptrophora japonica 265 1 PennatµLacea Sessiliflorae Kophobelem nidae Sclerobelemnon Sclerobelemnon theseus 257 1 Average 236

50

Appendix E Morphological matrix: 43 discrete characters are shown. See fig. 2.1 for characters description.

Spp./Character 1i' 2 2i 2' 2i' 3 3a 3b 3i 3' 3'b 3'a 3i' 4 4a 4b 4c 4i 4' 4'c 4'b 4'a 4i' 5 5a 5b 5c 5d 5e 5i 5' 5e' 5d' 5c' 5'b 5'a 5i' 6 6a 6i 6a' 6' 6i' El51557B3 2 2 3 2 2 7 2 1 2 8 1 0 1 5 4 0 0 3 3 0 0 2 5 7 2 4 2 2 0 2 5 0 1 2 2 2 1 2 0 8 0 2 3 ElA706B1 1 6 4 5 5 6 0 2 4 6 2 0 4 7 2 2 1 5 3 0 0 0 3 4 2 2 0 4 1 3 4 0 4 1 3 4 6 2 0 5 0 2 2 EpB178B2 2 2 3 2 2 8 2 2 2 9 3 0 3 4 4 0 0 3 2 0 0 2 6 7 2 4 3 2 0 2 5 0 1 3 2 2 1 9 1 2 7 9 3 EpB178B1 1 2 4 2 4 2 0 0 5 2 0 1 3 7 1 1 4 9 9 4 1 4 5 8 2 6 3 2 0 2 4 0 1 3 1 1 1 2 0 2 0 2 2 EpB178B3 2 2 5 2 2 8 2 1 2 8 0 0 2 4 4 0 0 3 2 0 0 2 4 8 1 5 3 2 0 2 6 0 1 3 3 0 6 5 1 9 0 3 6 EpB178B5 1 7 7 7 1 8 2 1 4 9 1 0 2 5 3 0 1 5 6 1 1 3 6 4 3 5 1 0 0 1 2 0 0 1 5 1 3 1 0 3 0 1 1 EpM141B3 1 6 4 5 5 6 2 2 1 7 2 2 2 8 5 1 2 3 7 2 1 2 8 5 7 2 0 1 0 3 2 0 0 1 2 1 3 6 1 4 2 5 4 EmP61B4 0 5 3 6 2 6 0 0 1 7 0 0 6 3 1 4 0 4 4 0 5 1 2 2 2 1 0 3 0 3 2 0 2 3 0 3 3 2 0 5 0 2 7 EpMI25B1 2 2 3 2 5 6 0 1 2 8 1 2 2 3 4 0 0 3 3 0 0 4 6 7 2 4 3 2 0 2 5 0 1 3 2 2 2 9 4 4 4 8 3 EpMI25B2 2 2 3 2 2 7 2 1 2 8 1 0 2 4 4 0 0 3 2 0 0 2 6 7 2 2 3 2 0 2 6 0 1 3 2 2 1 9 4 4 4 8 3 EpOC26B2 1 6 4 5 2 6 0 0 1 7 0 0 2 3 3 0 0 9 5 0 1 4 3 2 1 3 0 0 0 6 2 0 2 0 1 2 6 1 0 5 0 1 2 EfA716B2 2 3 3 3 4 5 0 0 2 5 0 0 2 4 3 2 0 3 3 0 2 2 9 9 1 5 1 3 3 1 8 3 3 0 1 4 1 5 2 4 3 5 5 EfP75Bu 2 3 3 3 5 4 0 0 2 4 0 0 3 4 3 2 0 2 3 0 2 2 9 9 0 5 1 3 3 1 8 3 3 0 1 3 1 5 2 4 3 5 5 EsPA98B1 2 3 3 3 1 5 0 2 2 4 0 0 3 4 3 0 0 2 3 0 0 2 8 8 2 4 3 3 2 2 6 2 3 3 3 0 1 8 3 3 3 5 7 Ep82140B1 2 2 3 2 2 7 2 1 2 8 1 0 1 5 2 4 0 3 3 0 2 2 5 7 2 4 2 2 0 2 5 0 1 2 2 2 1 2 0 8 0 2 3 EsuO28B3 2 2 6 7 7 4 1 0 2 4 0 1 0 6 1 1 2 4 7 2 1 3 8 5 7 2 0 1 0 3 2 0 0 1 2 1 3 6 1 4 2 5 4 EsuO32B2 2 3 3 3 4 5 0 1 2 5 1 0 2 4 3 0 0 7 3 0 0 2 9 9 5 5 2 3 2 4 9 4 3 2 4 7 1 7 0 4 2 7 4 EsuO32B5 1 6 4 5 2 6 0 0 1 7 0 0 5 4 3 1 0 4 5 0 3 2 2 3 1 2 0 3 2 3 2 2 0 4 2 1 4 1 0 5 0 1 2 EsuO32B6 2 4 2 4 3 7 4 0 2 5 0 0 2 6 0 3 0 2 8 0 2 4 1 0 0 0 0 0 0 2 0 0 0 0 0 0 7 0 0 0 0 0 0 Esu51555B1 2 2 3 2 2 7 2 1 2 8 1 0 1 5 4 0 0 3 3 0 0 2 5 7 2 4 2 2 0 2 5 0 1 2 2 2 1 2 0 8 0 2 3 Esu80836B2 2 2 5 2 4 4 0 2 2 4 2 0 3 9 3 2 1 1 9 1 2 4 4 9 6 4 0 3 1 3 9 4 3 1 7 8 1 9 2 1 2 7 2 984Efle 2 2 3 2 3 4 0 0 3 5 1 0 2 6 3 1 0 6 5 0 1 2 3 7 2 1 3 2 2 2 3 1 2 0 1 0 3 1 0 6 0 1 1 983Efus 2 3 3 3 4 6 0 0 3 6 0 0 2 4 3 2 0 3 1 0 0 2 9 9 0 0 4 3 3 4 9 3 3 2 2 4 1 3 0 1 0 3 5 732Elac 2 3 3 3 4 6 0 0 1 6 0 0 2 5 3 0 0 5 3 0 0 2 9 9 3 0 5 3 4 3 8 3 3 4 2 2 1 5 0 4 2 5 5 980Elax 2 2 3 2 3 5 0 0 2 6 1 0 2 4 3 0 0 8 3 0 0 2 7 5 1 3 3 2 0 2 3 0 1 3 0 1 1 5 1 6 1 4 3 977Epal 2 2 3 2 4 4 0 0 2 5 1 0 2 5 3 2 0 7 4 0 2 2 6 5 0 1 3 2 0 1 4 0 1 3 1 2 6 1 0 6 0 1 3 535Ecav 2 2 3 2 2 2 0 0 5 1 0 0 2 1 1 2 0 2 1 0 3 0 3 6 0 0 2 2 0 2 7 0 1 2 6 6 1 2 0 9 0 2 3 540Esin 2 2 3 2 2 2 0 0 5 1 0 0 2 1 1 2 0 2 1 0 3 0 3 6 0 0 2 2 0 2 8 0 1 2 6 6 5 3 0 7 0 3 3 526Lv ir 2 2 3 2 5 4 0 0 4 4 0 0 2 8 4 1 0 3 7 1 0 2 5 7 2 4 3 2 0 2 5 0 1 3 2 2 4 2 0 5 0 2 1 538Mba y 2 3 1 3 5 9 5 0 2 9 0 2 2 8 1 3 0 1 9 0 6 1 5 6 0 0 3 2 0 2 7 0 1 3 4 3 3 2 0 7 0 2 2 537Mfla 2 3 1 3 5 5 1 0 2 5 0 1 2 9 1 3 1 2 9 1 4 1 3 6 0 2 2 2 0 2 7 0 2 2 6 2 1 3 0 7 0 3 1 531Pste 2 2 3 2 5 5 1 2 2 5 2 1 2 9 4 1 1 1 7 0 0 2 5 7 2 4 3 2 0 2 5 0 1 3 2 2 4 1 0 6 0 1 1 535Pfla 2 2 2 2 5 6 0 0 1 8 1 0 2 7 1 3 0 6 8 0 5 1 6 3 0 1 1 3 0 7 3 0 3 1 1 3 4 1 0 6 0 1 1 536Ppla 2 2 2 2 5 6 0 0 1 8 1 0 2 7 1 3 0 6 8 0 5 1 6 3 0 1 1 3 0 7 3 0 3 1 1 3 4 1 0 6 0 1 1 974Phom 0 8 5 8 4 6 2 0 4 7 0 2 1 4 2 2 0 4 3 0 0 3 7 1 3 0 0 0 0 6 1 0 0 0 0 2 2 2 0 5 0 2 0 737Pkun 1 9 5 8 7 5 1 2 4 5 2 1 5 2 2 0 0 5 4 0 1 3 2 1 1 0 0 0 0 8 1 0 0 0 0 1 4 7 2 2 2 6 5 680Pnut 2 2 3 2 3 4 0 0 3 5 1 0 2 6 3 1 0 6 5 0 1 2 5 6 1 3 2 2 0 2 3 0 1 2 0 1 3 1 0 6 0 1 1 973Pcru 0 9 5 8 6 5 0 0 4 6 0 1 4 4 2 2 0 4 3 0 0 3 7 1 3 0 0 0 0 4 1 0 0 0 0 5 2 2 0 5 0 2 0 534Thar 2 2 3 2 3 5 0 0 2 6 0 1 2 4 3 0 0 8 3 0 0 2 7 5 1 3 3 2 0 2 3 0 1 3 0 1 1 5 1 6 1 4 2 GvA679 2 2 3 2 2 7 2 1 2 8 1 0 2 4 4 0 0 3 2 0 0 2 6 7 2 4 3 2 0 1 5 0 1 3 2 2 1 9 5 4 6 8 4 GfB1B2 2 3 1 3 4 9 5 0 2 9 0 2 2 8 1 3 0 1 9 0 6 1 2 0 0 0 0 0 0 5 1 0 0 0 0 4 6 7 2 2 0 4 0 GfB1B3 2 3 1 3 4 9 5 0 2 9 0 2 2 8 1 3 0 1 9 0 6 1 5 6 0 0 3 2 0 2 7 0 0 3 4 2 1 1 0 1 0 1 6 GvN213B6 2 2 3 2 2 7 2 1 2 8 1 0 2 4 4 0 0 3 2 0 0 2 6 7 2 4 2 2 0 2 5 0 1 2 2 2 1 6 2 6 0 4 3 GmB108B1 1 2 2 2 4 4 0 0 2 5 0 2 2 6 3 1 0 7 5 0 1 2 6 4 1 3 2 3 0 2 2 0 3 2 0 1 3 1 0 1 0 1 1 GmB108B2 2 2 4 2 2 8 3 2 2 7 0 1 2 2 0 1 0 5 2 0 0 1 6 7 2 4 2 4 0 2 5 0 3 2 2 2 1 1 0 2 0 1 5 GmB127B2 2 3 1 3 6 5 1 0 2 5 0 1 1 6 1 3 1 4 5 1 2 1 6 7 0 2 1 2 1 7 7 1 2 1 2 2 2 1 0 6 0 1 3 521Gfla 2 2 3 2 2 7 2 1 2 8 1 0 2 4 4 0 0 2 2 0 0 2 6 7 2 4 3 2 0 2 5 0 1 3 2 2 1 9 4 4 4 8 3 523Gmar 2 2 3 2 3 8 2 1 3 8 1 0 3 5 4 0 0 1 3 0 0 2 6 7 2 4 3 2 0 2 5 0 1 3 2 2 1 9 5 4 5 8 3 522Gven 2 2 3 2 2 7 2 1 2 8 1 0 2 4 4 0 0 3 2 0 0 2 6 7 2 4 3 2 0 2 5 0 1 3 2 2 1 9 4 4 4 8 3 Pb13B1 2 2 3 2 5 3 0 0 2 2 0 0 2 5 1 1 1 1 6 2 0 2 5 7 2 2 1 2 2 2 5 1 0 1 2 2 1 4 0 2 4 6 3 Pb25P14 2 2 3 2 4 3 0 0 2 3 0 0 1 5 1 1 1 1 6 2 0 2 5 7 2 4 1 3 2 2 5 1 3 1 2 3 1 5 0 2 1 5 3 PaP71 1 1 4 1 3 3 1 0 3 2 0 1 2 4 4 0 0 3 2 0 0 2 6 7 2 4 2 2 0 2 6 0 2 2 2 2 1 2 0 8 0 2 3 Pb1 2 2 3 2 4 3 0 0 2 3 0 0 1 4 3 1 0 6 3 0 1 2 5 7 2 4 3 2 0 2 5 0 1 3 2 2 1 4 0 1 4 6 4 Pb53 2 2 3 2 4 3 0 0 2 3 0 0 1 5 1 1 1 1 6 2 0 2 6 7 2 4 3 2 0 2 5 0 1 3 2 2 2 4 0 2 5 7 3 PbP3288 2 2 3 2 4 5 2 0 2 4 1 0 2 7 2 4 3 6 4 3 0 2 7 7 2 0 3 2 0 2 7 0 1 3 5 3 2 6 0 1 1 5 3 PbA471 2 6 1 6 2 6 1 1 3 7 1 2 2 4 4 0 0 3 2 0 0 2 4 8 1 2 3 3 2 2 8 4 1 1 5 8 1 8 2 2 0 5 1 PrA511 1 3 3 3 1 7 2 1 2 8 1 0 1 5 4 0 0 3 3 0 0 2 5 7 2 4 2 2 0 2 5 0 1 2 2 2 1 2 0 8 0 2 3 PrA538 2 2 3 2 3 7 2 1 2 8 1 0 1 5 4 0 0 3 3 0 0 2 5 7 2 4 2 2 0 2 5 0 1 2 2 2 1 2 0 8 0 2 3 Pb40B 1 3 3 3 4 3 0 0 2 3 0 0 2 4 1 1 1 1 5 2 0 2 7 6 2 3 5 1 0 2 5 0 2 4 1 2 2 4 0 2 5 7 3 Pb41B 2 2 3 2 5 1 0 0 6 1 0 0 4 2 1 0 0 3 6 0 1 5 6 7 1 4 6 2 0 2 5 0 1 5 2 1 2 2 0 7 0 2 3 Pb22B3 2 2 3 2 4 3 0 0 2 3 0 0 2 6 1 1 2 1 4 1 1 1 3 6 2 0 0 0 0 5 6 0 0 0 2 1 3 4 0 1 4 6 3 Pb7B1 2 3 2 3 3 3 0 0 3 3 0 0 1 6 2 1 3 2 6 3 1 2 5 5 2 1 4 0 0 6 6 0 2 4 1 2 2 1 0 3 0 1 0 Pb23B2 2 2 3 2 5 3 0 0 2 2 0 0 2 5 1 1 1 1 6 2 0 2 5 8 4 4 3 2 0 2 5 0 1 3 2 2 1 3 0 1 4 5 4 Pb20B1 2 2 3 2 4 3 0 0 2 3 0 0 1 5 2 1 1 1 5 2 0 2 6 7 2 4 3 2 0 2 5 0 1 3 2 2 2 3 0 4 4 5 3 Pb47B1 2 2 3 2 4 3 0 0 2 3 0 0 2 4 2 1 1 1 4 2 0 2 6 6 2 4 3 2 0 6 4 0 1 3 2 2 1 4 0 1 4 6 3

51 Pb6B3 2 2 3 2 4 3 0 0 2 3 0 0 5 5 1 1 1 1 6 2 0 2 5 7 2 4 2 2 0 2 6 0 1 2 2 3 2 4 0 5 0 4 3 Pb20B3 2 2 3 2 4 3 0 0 2 3 0 0 2 4 1 1 1 1 4 2 0 1 7 7 2 4 3 2 0 2 5 0 1 3 2 2 2 4 0 1 4 6 3 Pb44B2 2 2 3 2 4 3 0 0 2 3 0 0 2 4 1 1 1 1 5 2 0 2 6 7 2 4 3 2 0 3 5 0 1 3 2 2 1 4 0 2 4 6 3 524Pbip 2 2 3 2 2 7 2 1 2 8 1 0 2 4 4 0 0 3 2 0 0 2 6 7 2 4 2 2 0 2 5 0 1 2 2 2 1 2 0 8 0 2 3 123Pbip 2 2 3 2 4 3 0 0 2 3 0 0 1 5 4 1 0 1 6 0 4 2 5 7 2 4 3 2 0 2 5 0 1 3 2 2 1 3 0 3 4 5 3 679Pcit 0 3 6 3 2 4 0 0 2 4 1 0 2 3 1 0 0 7 3 0 0 1 7 4 3 3 2 0 0 2 3 0 0 2 3 2 4 2 0 5 0 2 1

52 Appendix F Changes done to convert number of nucleotides to a discrete character.

53