Mitochondrial Genome Architecture in Sponges, With

MITOCHONDRIAL GENOME ARCHITECTURE IN SPONGES, WITH

DESCRIPTIONS OF TWO NEW SPECIES FROM THE ABYSSAL

NORTHEAST PACIFIC

A Thesis

Presented to the

Faculty of the

Moss Landing Marine Laboratories

California State University Monterey Bay

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Marine Science

Amanda Suzanne Kahn

Fal12010 111

Amanda Suzanne Kahn

For those who patiently support me while I pursue my dreams. v

ABSTRACT

Mitochondrial Genome Architecture in Sponges, with Descriptions of Two New Species from the Abyssal Northeast Pacific by Amanda Suzanne Kahn Master of Science in Marine Science California State University Monterey Bay, 2010

Mitochondrial genomics is an emerging field that aims to characterize evolution of the mitochondrion, an organelle found in nearly all eukaryotes. Such a ubiquitous feature presents a platform on which to address questions about phylogenetics, evolution, and molecular mechanisms across taxa. Porifera is a diverse phylum containing animals believed to be the first true multicellular organisms, yet few mitochondrial genomes have been sequenced across groups within the phylum. Classes within Porifera have parallel structures to those found in animals but not found in other sponge groups; therefore, relationships among and within classes are important for our understanding of the evolution of the phylum. Here I present two nearly complete mitochondrial genomes of two hexactinellid sponges, bringing the total number up to five. The five genomes provide coverage at various taxonomic levels within Hexactinellida, allowing a large range of comparisons of features such as gene loss, changes in gene order, and transcriptional and translational mechanisms. I also evaluate the phylogenetic value of mitochondrial gene sequences at different taxonomic levels. Finally, the two species, Bathydorus laniger and Docosaccus maculatus, are new to science and are formally described. Both are found in the same region and have similar gross morphology, but are from two different families within the order Lyssacinosida. Vl

TABLE OF CONTENTS PAGE ABSTRACT ...... V LIST OF TABLES ...... VIII LIST OF FIGURES ...... IX ACKNOWLEDGEMENTS ...... XI INTRODUCTION ...... 1 References ...... 3 1 MITOCHONDRIAL GENOME EVOLUTION IN GLASS SPONGES ...... 5 Abstract ...... 5 Introduction ...... 6 Methods ...... 8 Phylogenetic analysis ...... 9 Results ...... 10 Genome structure and composition ...... 10 Protein-coding genes ...... 12 Frameshifting insertions ...... 12 Gene arrangement ...... 13 Phylogenetic analysis ...... 13 Discussion ...... 15 Genome structure and composition ...... 15 Testing for linearity of the genome ...... 16 Protein-coding genes ...... 18 Translational frameshifts ...... 18 Gene arrangement ...... 19 Phylogenetic analysis ...... 20 Conclusions ...... 21 References ...... 22 2 BATHYDORUS LANIGER AND DOCOSACCUS MACULATUS (L YSSACINOSIDA; HEXACTINELLIDA): TWO NEW SPECIES OF GLASS SPONGES FROM THE ABYSSAL EASTERN NORTH PACIFIC OCEAN ...... 26 Abstract ...... 26 Vll

Introduction ...... 27 Material and Methods ...... 28 Results ...... 30 Bathydorus laniger sp. n ...... 30 Material examined ...... 30 Diagnosis ...... 30 L yssacine framework ...... 31 Spicules ...... 33 Etymology...... 34 Remarks ...... 34 Docosaccus maculatus sp. n ...... 36 Material examined ...... 36 Diagnosis ...... 36 Description ...... 36 Lyssacine framework ...... 37 Spicules ...... 39 Etymology...... 42 Remarks ...... 42 Discussion ...... 42 References ...... 44 A GENBANK ACCESSION ENTRIES FOR BATHYDORUS LANIGER AND DOCOSACCUS MACULATUS ...... 46 B GLOSSARY OF TERMS USED IN HEXACTINELLID TAXONOMY ...... 64 C MEASUREMENTS OF SPICULES FOUND IN BATHYDORUS LANIGER ...... 68 D MEASUREMENTS OF SPICULES FOUND IN DOCOSACCUS MACULATUS ...... 70 Vlll

LIST OF TABLES

PAGE

Table 1-1. Comparison of protein-coding genes found in five species of glass sponges, and the pairwise similarities of their nucleotide and amino acid sequences ...... 11 Table 1-2. Variations in the trnY(gua) repeats found in the otherwise non-coding region between nadl and cob of Docosaccus maculatus. All copies were 65 bp long. The first copy was at the 5' end of the repetitive region, the third copy at the farthest 3' end; the bold, second sequence is used as a reference sequence since it was predicted to be the functional trnY gene by tRNAscan- SE (Lowe and Eddy 1997) ...... 12 Table 2-1. Spicule dimensions of Bathydorus laniger sp. n., from Station M, California, USA (dimensions in ~J.m, except for the choanosomal and pros tal diactin lengths, which are in mm) ...... 35 Table 2-2. Spicule dimensions of Docosaccus maculatus sp. n., from Station M, California, USA (dimensions in ~J.m) ...... 40 IX

LIST OF FIGURES

PAGE

Figure 1-1. The largest non-coding region (1,439 base pairs) in Docosaccus maculatus was a repetitive region between nadl and cob. The region contained stuttering repeats of the trn Y gene and the 5' end of cob (gray squares). Green squares indicate coding genes, tan represent tRNA genes, and the thin line represents the non-coding region ...... 12 Figure 1-2. Gene order in the mitochondrial genomes of five species of glass sponges. Green - protein-coding genes, red - ribosomal RNA genes, tan - tRNA, with each gene identified by the single-letter IUPAC code of the amino acid for which it codes. Multiple serine and leucine residues are differentiated as S1:trnS(uga), S2:trnS(ucu), L1:trnL(uag), ~:trnL(uaa), L3:trnL(cag). Non coding regions are not displayed; figure is not to scale. Data for A. vastus from Rosengarten et al. (2008), data for I. panicea and S. nux from Haen et al. (2007) ...... 13 Figure 1-3. Evolutionary relationships of9 taxa based on consensus of 12 consensus trees from protein-coding genes. Evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei 1987). The consensus tree was assembled from consensus trees of each of 12 genes: atp6, atp9, cob, cox], cox2, cox3, nadl, nad2, nad3, nad4, nad4L, and nad5, and the percentage of individual gene trees in which the associated taxa clustered together are shown next to the branches. Each consensus tree was inferred from 10,000 bootstrap replicates (Felsenstein 1985). Evolutionary distances were computed using the F84 substitution model (Felsenstein 1984). All positions containing gaps and missing data were eliminated from the dataset. Phylogenetic analyses were conducted in SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs within the Phylip package (Felsenstein 1989) ...... 14 Figure 1-4. Evolutionary relationships of9 taxa based on a concatenation of 12 protein-coding genes; there were a total of 10,704 positions in the final data set. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei 1987). The bootstrap consensus tree inferred from 10,000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 0,000 replicates) are shown next to the branches (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion X

option). Phylogenetic analyses were conducted in MEGA4 (Tamura et al. 2007) ...... 15 Figure 1-5. 1.2% agarose gel prepared with sodium boric acid, run at 180V for 18 minutes. The first well contains the PCR product from a long PCR between nad6 and rns on DNA extracted from Docosaccus maculatus. The second well is a negative control, and the third is the DNA size ladder...... 18 Figure 1-6. Cladogram representing some major changes in mitochondrial gene arrangement with phylogenetic context. Red bars indicate losses of genes or frameshifts (although the loss of nad6 may be a shift into the unsequenced region between nad4 and coxl), blue bars indicate gene shifts, and green bars indicate+ 1 frameshifts caused by tryptophan (UGG) codons ...... 20 Figure 2-1. Collection location of two new species of glass sponges with a unique plate-like morphology: Station M (4,100 m depth, 34°50'N, 123°0'W), a long- term abyssal study site in the northeast Pacific ...... 28 Figure 2-2. Bathydorus laniger sp. n. holotype, whole body images. A. In situ photograph of whole organism taken by ROV Tiburon. Atrial surface with marginal prostalia visible. Credit: MBARI. B. Dermal surface, with pleural prostalia (P) seen protruding as long, single diactins. Scale bar: 10 em ...... 31 Figure 2-3. Bathydorus laniger sp. n. from Station M, California, USA; scale diagram of spicule arrangement. The atrial surface is at the top of the image while the dermal surface is at the bottom; prostal diactins are omitted. Scale bar: 200 /liD ...... 32 Figure 2-4. Bathydorus laniger sp. n., spicule images from SEM. A. Dermal stauractin. B. Atrial hexactin. C. Hypodermal pentactin. D. Tip of choanosomal diactin. E. Oxyhexaster, oxyhemihexasters, and oxyhexactin. Scale bar: 200 11m ...... 34 Figure 2-5. Docosaccus maculatus sp. n., whole body images ofholotype. A. In situ photograph taken by ROV Tiburon. Atrial surface with black and white spots visible. Black spots are caused by shadows in holes that perforate the body (parietal oscula), white spots occur where anchoring spicule tufts are visible through the body. Credit: MBARI. B. Dermal surface, showing small papillae consisting of anchoring spicule bundles. Scale bar: 5 em ...... 37 Figure 2-6. Diagram of spicule arrangement for Docosaccus maculatus sp. n. from Station M, California, USA. Scale bar: 200 /liD ...... 38 Figure 2-7. Spicule images of Docosaccus maculatus sp. n. (LM). A. Atrial pentactin. B. Choanosomal hexactin. C. Dermal hexactin. Scale bar (between Figures 7a and 7b): 200 /liD. D-G. Oxyhexasters. Scale bar (between 7d and 7f): 50 /liD ...... 41 Figure 2-8. Floricomes of Docosaccus maculatus sp. n. (SEM). A. Complete floricome. Scale bar: 50 /liD. B. Close-up offloricome secondary rays. Scale bar: 10 /liD ...... 41 Xl

ACKNOWLEDGEMENTS

I would like to first thank Drs. Jonathan Geller, Greg Cailliet, and Ken Smith, for being a supportive and constructive thesis committee. Jacob Ellena, Mike Vardaro, Henry Ruhl, the crew of the R/V Western Flyer, and pilots of the ROV Tiburon were responsible for collection of sponge specimens necessary for the species descriptions. Dr. Henry Reiswig taught me the ins and outs ofhexactinellid sponge taxonomy, provided many reprints of old taxonomic literature, and assisted with SEM work at the University of Victoria. Sara Tanner led me through SEM work at MLML. Drs. Reiswig, Dorte Janussen, and Konstantin Tabachnick provided valuable, educational comments that greatly improved the taxonomic descriptions. I acknowledge Lynn McMasters for her assistance with digital figures. Kristin Meagher assisted with data collection and spicule measurements. Clark Pennelly supported me while writing this document. I would also like to thank my funding sources-the donors to the James Nybakken Scholarship, the David and Lucile Packard Research and Travel Grant, the Marine Technology Society, and ship time from Dr. Ken Smith and the Monterey Bay Aquarium Research Institute-for supporting this research. Finally, I thank my mother, Suzanne Kahn, for all of her support and encouragement to pursue my interests, and for patiently allowing me to do so for many years. 1

INTRODUCTION

Mitochondrial genomics aims to characterize and compare properties of mitochondrial genomes-organelles that are found in nearly all eukaryotes (Burger et al. 2003). Based on a recent NCBI Genome search, over 2,000 mitochondrial genomes have been sequenced on eukaryotes ranging from bacteria to fungi, plants, and animals. Most animal genomic data come from bilaterians. Few mitochondrial genomes have been sequenced from non-bilaterian animals (sponges, placozoans, cnidarians, and ctenophores); however, comparisons of mitochondrial genomes among these groups may contribute to hypotheses regarding the origin of metazoans (King et al. 2008), relationships among non-bilaterian taxa (Philippe et al. 2009), and relationships within taxa, including the phylum Porifera (Lavrov et al. 2008). Phylogenies for the phylum Porifera, the sponges, have historically been based on morphological similarities (e.g. Reiswig 2006) that rely on interpretation of morphological characters including gross morphology, spicule types and size ranges, structure of the aquiferous system, and skeletal arrangement (Bergquist 1978, Hooper and Van Soest 2002, Lee et al. 2007). Interpretation of these morphological characters has led to disagreement between systematists and numerous changes to sponge phylogenies through time (Bergquist 1978, Hooper and Van Soest 2002). Molecular phylogenies use genetic information instead of, or in addition to, morphological characters. Although methods vary, all are based on mutations that accumulate over time, including point mutations, insertions, deletions, and nucleotide rearrangements (Campbell and Reece 2002). For example, each nucleotide position in a genome has one of four possible character states: adenine, thymine, cytosine, or guanine. With four character states at each nucleotide position, the potential number of differences between taxa can be much greater (4n, with n being the number ofbases examined); thus the resolution of molecular phylogenies can be greater than that of morphological phylogenies. Unfortunately, molecular phylogenies for the phylum Porifera have thus far had low resolution and are discordant with morphological characters (i.e. Borchiellini et al. 2001, Erpenbeck and Worheide 2007, Sperling et al. 2009). Loci commonly used for 2

phylogenetic analysis in other taxa, such as ISS ribosomal regions in the mitochondrial genome, mutate too slowly to produce the necessary resolution to differentiate between higher taxonomic levels ofPorifera such as families, orders, and classes. A multi-gene phylogeny integrates the different rates of mutation among genes and produces a more robust view of systematic relationships (Pick et al. 201 0). In addition to uses in molecular phylogenetics, mitochondrial genomes provide molecular features that can function as distinct characters for comparisons between groups. Differences in the structure of the genome such as gene order (Lavrov and Lang 2005), gene loss (Lavrov et al. 2008, Wang and Lavrov 2008), and changes in transcriptional or translational mechanisms (Haen et al. 2007) have been observed in many groups (Burger et al. 2003). These features may be useful as characters in phylogenies, but the taxonomic levels at which such characters change is not known. Before this study, nearly complete mitochondrial genomes of three hexactinellid species were available. Chapter 1 introduces the nearly complete mitochondrial genomes of two more species, providing opportunity for phylogenetic studies based on gene sequences and mitochondrial genome evolution. The five genomes provide coverage at various taxonomic levels within Hexactinellida, allowing the greatest range of comparisons. Aphrocallistes vastus Schulze, 1886 (Hexactinosida; Aphrocallistidae) and Iphiteon panicea Bowerbank, 1869 (Hexactinosida; Dactylocalycidae) are in different families within the class Hexactinosida. Sympagella nux Schmidt, 1870 (Lyssacinosida; Rossellidae) is in the class Lyssacinosida, and will be joined with the two new, nearly complete genomes from this study. Bathydorus laniger n. sp. falls within the same family as S. nux, but is in a different subfamily while Docosaccus maculatus n. sp. (Lyssacinosida; Eupectellidae) is in a different family from S. nux and B. laniger. These two hexactinellid species under study are new to science. The sponges are morphologically very similar, but analysis of their spicules places them in two different families within Order Lyssacinosida. Chapter 2 features descriptions of Bathydorus laniger, a plate-like sponge with marginal prostalia radiating from the peripheral edge of the sponge, and Docosaccus maculatus, another plate-like sponge with tufts of anchoring prostalia projecting down into the sediments. 3

REFERENCES

Bergquist, P.R. (1978). Sponges. University of California Press, Berkeley, CA. 267 pp.

Borchiellini C., M. Manuel, E. Alivon, N. Boury-Esnault, J. Vacelet, andY. Le Parco (2001). Sponge paraphyly and the origin of Metazoa. Journal ofEvolutionary Biology, 14:171-179. Burger, G., M. W. Gray, and B. F. Lang (2003). Mitochondrial genomes: anything goes. TRENDS in Genetics, 19( 12): 7 09-716.

Campbell, N. A., and J. B. Reece (2002). Biology, 61h edition. Benjamin Cummings, San Francisco, CA. 1247 pp. Erpenbeck D., and G. Worheide (2007). On the molecular phylogeny of sponges (Porifera). Zootaxa, 1668:107-126. Haen K. M., B. F. Lang, S. A. Pomponi, and D. V. Lavrov (2007). Glass sponges and bilaterian animals share derived mitochondrial genomic features: a common ancestry or parallel evolution? Molecular Biology and Evolution, 24(7): 1518- 1527. Hooper, J. N. A., and R. W. M. Van Soest (Eds.) (2002). Systema Porifera: A Guide to the Classification of Sponges. Kluwer Academic/Plenum Publishers, New York. 1810 pp. King, N., M. J. Westbrook, S. L. Young, A. Kuo, M. Abedin, J. Chapman, S. Fairclough, U. Hellsten, Y. Isogai, I. Letunic, M. Marr, D. Pincus, N. Putnam, A. Rokas, K. J. Wright, R. Zuzow, W. Dirks, M. Good, D. Goodstein, D. Lemons, W. Li, J. B. Lyons, A. Morris, S. Nichols, D. J. Richter, A. Salamov, JGI Sequencing, P. Bork, W. A. Lim, G. Manning, W. T. Miller, W. McGinnis, H. Shapiro, R. Tjian, I. V. Grigoriev, and D. Rokhsar (2008). The genome of the choanoflagellate Monosiga brevicollis and the origin ofmetazoans. Nature, 451:783-788. Lavrov D. V., and B. F. Lang (2005). Poriferan mtDNA and animal phylogeny based on mitochondrial gene arrangements. Systematic Biology, 54(4):651-659. Lavrov D. V., X. Wang, and M. Kelly (2008). Reconstructing ordinal relationships in the Demospongiae using mitochondrial genomic data. Molecular Phylogenetics and Evolution, 49:111-124. Lee, W. L., D. W. Elvin, and H. M. Reiswig (2007). The Sponges of California: A guide and key to the marine sponges of California. Monterey Bay Sanctuary Foundation, U.S. A. 130 pp. Philippe, H., R. Derelle, P. Lopez, K. Pick, C. Borchiellini, N. Boury-Esnault, J. Vacelet, E. Renard, E. Houliston, E. Queinnec, C. DaSilva, P. Wincker, H. Le Guyader, S. Leys, D. J. Jackson, F. Schreiber, D. Erpenbeck, B. Morgenstern, G. Worheide, 4

and M. Manuel (2009). Phylogenomics revives traditional views on deep animal relationships. Current Biology, 19:706-712. Pick, K. S., H. Philippe, F. Schreiber, D. Erpenbeck, D. J. Jackson, P. Wrede, M. Wiens, A. Alie, B. Morgenstern, M. Manuel, and G. Worheide (2010). Improved phylogenomic taxon sampling noticeably affects non-bilaterian relationships. Molecular Biology and Evolution, 27(9):1983-1987. Reiswig, H. M. (2006). Classification and phylogeny ofHexactinellida (Porifera). Canadian Journal ofZoology, 84: 195-204. Sperling, E. A., K. J. Peterson, and D. Pisani (2009). Phylogenetic-signal dissection of nuclear housekeeping genes supports the paraphyly of sponges and the monophyly ofEumetazoa. Molecular Biology and Evolution, 26(10):2261-2274. Wang, X., and D. V. Lavrov (2008). Seventeen new complete mtDNA sequences reveal extensive mitochondrial genome evolution within the Demospongiae. PLoS ONE, 3(7):1-11. 5

CHAPTER!

MITOCHONDRIAL GENOME EVOLUTION IN GLASS SPONGES

Amanda S. Kahna, *, Jonathan B. Gellera aMoss Landing Marine Laboratories, 8272 Moss Landing Road, Moss Landing, CA 95039, USA *corresponding author. E-mail address: [email protected] (A.S. Kahn)

ABSTRACT I have sequenced nearly complete mitochondrial genomes of two new species of glass sponges, Bathydorus laniger (Lyssacinosida; Rossellidae) and Docosaccus maculatus (Lyssacinosida; Euplectellidae), contributing taxonomic diversity to the current collection ofhexactinellid mitochondrial genomes and bringing the number of genomes sequenced up to five. Mitochondrial DNA was sequenced using a combination of standard PCR, long PCR, restriction digestion and cloning, and primer walking. Coding regions were found using bioinformatics software and annotated for analysis. A single, contiguous region of over 15,000 bp from each genome was sequenced stretching from the gene coding for Cytochrome c oxidase subunit 1 to NADH Dehydrogenase subunit 4. Gene size and content did not vary significantly across all of the five hexactinellid taxa, but several unique structural variations were found in comparisons of hexactinellid taxa with demosponge and bilaterian taxa. Gene rearrangements were common, especially movements of transfer RNA genes. Single nucleotide insertions disrupted coxl in B. laniger and nad2 in D. maculatus, either rendering the genes non functional or requiring post-transcriptional editing or translational repair. Repetitive elements also added complexity to the genomes. Phylogenies from these data support the mon9phyly ofHexactinellida and suggest re-examination of the taxonomic placement of the family Dactylocalycidae. 6

INTRODUCTION The first complete mitochondrial genome sequenced was the human genome in 1981 (Anderson et al. 1981 ). Subsequent genomes isolated from other animals led to a paradigm of"genetic economy" ofbilaterian mitochondrial DNA (Attardi 1985): in this paradigm, all animal mitochondria harbor a common set of genes that encode for enzymes used in oxidative phosphorylation, two subunits of ribosomal RNA, and transfer RNAs for translating those genes (Boore 1999), and the space surrounding those genes has been pruned down so that repetitive regions, introns, and other non-coding portions of the DNA have been removed. As full genome sequencing became more tractable and genomes were sequenced from non-bilaterian and non-animal sources, this paradigm did not carry over. Instead, there was evidence of repetitive regions, modified tRNA and rRNA structures, non-coding sequences, and introns (Burger et al. 2003). Variations in these architectural structures of the mitochondrial genome can be used to infer phylogenetic relationships (Bridge et al. 1992, 1995; Boore et al. 1995, Boore and Brown 1998), just as morphological features of body plans are. Over a thousand animal mitochondrial genomes have now been sequenced; however, most ofthese genomes come from bilaterian animals (Lavrov 2011) that exemplify the paradigm of"genetic economy" (Attardi 1985). The structure of the mitochondrial genome of the choanoflagellate Monosiga brevicollis, a group closely related to the Eumetazoa, differs greatly in size, gene composition, presence of introns, and even in number of genes encoded (Lang et al. 2002), lending itself more to the idea, that, in mitochondrial DNA, "anything goes" (Burger et al. 2003). Non-bilaterian groups, such as sponges and cnidarians, lie between choanoflagellates and bilaterians, and so they may hold evidence of the transition from the large genomes of choanoflagellates to the compact ones ofbilaterians (Lavrov 2011). The complete genomes of four placozoans (Dellaporta et al. 2006, Signorovitch et al. 2007), cnidarians from all major classes (Kayal and Lavrov 2008; Lavrov 2011), and several taxa of demosponges and homoscleromorph sponges (Wang and Lavrov 2007, 2008) cover some non-bilaterian groups, but only three genomes have been sequenced from hexactinellid sponges (Haen et al. 2007, Rosengarten et al. 2008) and none have been sequenced from calcareous sponges nor from ctenophores. 7

A thorough study ofthe mtDNA ofdemosponge taxa verified that five major clades exist (Wang and Lavrov 2008). Changes in gene order and coding direction were the most common variations between the groups, and in one group, the GO (homoscleromorphs), introns were even found (Wang and Lavrov 2007). With such differences present within the Demospongiae, adequate taxonomic coverage is important to understanding changes in the mitochondrial genomes. I have sequenced the nearly complete mitochondrial genomes of two species of glass sponges, bringing total coverage up to five genomes in Class Hexactinellida. I selected Bathydorus laniger, a species of glass sponge within Family Rossellidae, because it is a member of the family of another species whose mitochondrial genome has been almost completely sequenced: Sympagella nux. The other genome sequence comes from Docosaccus maculatus, a species of sponge within Family Euplectellidae. This sequence allows a comparison from a different family within the same order as the two rossellids (Order Lyssacinosida). Bathydorus laniger and Docosaccus maculatus are of various phylogenetic distances from other hexactinellid mitochondrial genomes that have been sequenced, allowing comparative studies of mitochondrial genome structure, evolution, and the rate at which structural changes occur. Comparative mitochondrial genomics commonly observes "genetic conservatism versus structural flamboyance" (Burger et al. 2003) conservation of gene function and sequence but several changes in gene order, genome structure, and transcriptional and translational mechanisms. The two genomes described here will contribute to such comparative studies among hexactinellids. In addition to evaluating genome architecture as characters in evolution, gene sequences from these two species can be used to infer phylogenies within the class Hexactinellida and within the phylum Porifera. Poriferan systematics has been reorganized frequently in the past (Reiswig 2006) and many relationships remain unresolved today. Sponges have few characters that are useful for taxonomic classifications: gross morphology and shape change with water conditions while color varies within a single individual. Successful programs such as the World Porifera Database (WPD; http://www.marinespecies.org/porifera/index.php), Sponge Barcoding Project (SBP; http://www.spongebarcoding.org/), and Poriferan Tree of Life project 8

(PorToL; http://homepage.uab.edu/thacker/portol.htm; Maddison and Schulz 2007) compile morphological and molecular data, with major goals of solidifying the systematics of sponge taxa. The molecular data from the two species presented here add support for a phylogeny that used mitochondrial sequence data from the 18S ribosomal RNA region to ascertain higher taxonomic relationships within the phylum (Dohrmann et al. 2009).

METHODS Specimens of both Bathydorus laniger and Docosaccus maculatus were collected by remotely operated vehicle (Dive Tl 094, ROV Tiburon) during a cruise to Station M, a long-term abyssal study site 200 km west ofPoint Conception, California, USA (4,100 m depth, 34°50'N, 123°0'W; Smith and Druffel1998). Intact sponges were gently collected using a Raptor manipulator arm and brought up slowly to the surface. Once on deck, sponge samples were collected and deep frozen immediately in liquid nitrogen and stored at -80°C until used. Samples remained frozen in liquid nitrogen until DNA was extracted using DNeasy Animal and Blood Tissue Extraction kits (Qiagen, USA). Sequencing the mitochondrial genomes relied on several approaches, including standard PCR, long PCR (Burger et al. 2007), and restriction digests and cloning. First, anchoring sequences within cox3, nad2, nad5, rnl, and rns were amplified using primers developed from conserved regions in the three other known hexactinellid mitochondrial genomes (Aphrocallistes vastus, NC_ 01 0769; Iphiteon panicea, EF537576.1; and Sympagella nux, EF537577.1). Standard PCR was performed using GoTaq master mix (Promega, Wisconsin, USA). With these and other anchoring regions, new template specific regions were designed for long PCR using Primer3Plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and guidelines reviewed by Abd-Elsalam (2003). The remaining portions of the genome, from coxl to nad4, were amplified in overlapping fragments 2,000-5,000 base pairs long using New England Biolabs LongAmp master mix and polymerase (New England Biolabs, Massachusetts, USA) using the following cycling conditions: a 94°C initial melting step, followed by 25 cycles of a 10 s melting step at 94°C, 60 s at a primer-specific annealing temperature (ranging from 45°C to 52°C), and an extension step of 60s per kb of expected 9

product at 65°C. The 25 cycles were followed by a final extension step of 65°C for 10 min. Read lengths for conventional Sanger sequencing are typically 400 to 600 base pairs (Fran<;a et al. 2002) and although they can be up to 1,000 base pairs long (Marziali and Akeson 2001 ), this is not sufficient for bidirectional sequencing of the long PCR products. In consequence, products were sequenced in one of two ways: by restriction digestion and by primer walking. Long PCR products were first concentrated using ethanol precipitation and were re-suspended in TE (10 mM Tris, 1 mM EDTA). Enzymes Alui, Rsai, and Haeiii (New England Biolabs, Massachusetts, USA) digested 5 11g oflong PCR products into smaller fragments of suitable lengths to be sequenced. Digested fragments were concentrated with ethanol precipitation again and were re-suspended in water. Digests from the three enzymes were mixed, blunted, cloned, and transformed according to protocols in the TOPO® Zero Blunt cloning kit and transformed into TOPO One-Shot® Competent Cells. Cells were incubated 1 hr, then plated and incubated at 3 7°C for 16 hr. Colonies were picked using a platinum wire into Lyse-and-Go (Pierce Protein Research Products, Illinois, USA) to prepare template for PCR using standard M13 primers. Colony PCR products were sent out for sequencing at Elim Biopharmaceuticals Inc (Hayward, CA). Gaps that remained following sequencing of restriction digests were filled using primer walking in small, overlapping fragments. Sequencing primers were designed using Primer3Plus and sequencing was carried out by Elim Biopharmaceuticals Inc. (Hayward, CA). Sequences were processed and assembled using Geneious software (Drummond et al. 201 0). Coding regions were identified by similarity to known genes using BLAST searches of GenBank, followed by examinations of gene translations in Geneious. Transfer RNA (tRNA) sequences were predicted using tRNAscan-SE program (Lowe & Eddy 1997, available online at http://lowelab.ucsc.edu/tRNAscan-SE/).

Phylogenetic analysis Phylogenetic analyses were conducted on protein-coding genes from Bathydorus laniger and Docosaccus maculatus. Protein-coding gene sequences were derived from GenBank files of the following taxa: hexactinellids Sympagella nux (EF537577), Iphiteon 10

panicea (EF537576), and Aphrocallistes vastus (NC _ 0 10769), demosponges Xestospongia muta (EU237490) and Ephydatia muelleri (EU237481), homoscleromorph Oscarella carmela (EF081250), and the choanoflagellate Monosiga brevicollis (NC_004309) as an outgroup. Each protein-coding gene (atp6, atp9, coxl, cox2, cox3, nadl, nad2, nad3, nad4, nad4L, and nad5) was aligned using ClustalW (Thompson et al. 1994, 2002), Alignments were bootstrapped 10,000 times using the SEQ BOOT program in PHYLIP (Felsenstein 1985). Distance matrices were calculated for each replicate alignment using the DNADIST program in PHYLIP (Felsenstein 1989). Finally, phylogenetic trees were constructed for each matrix using the Neighbor-Joining method ofSaitou and Nei (1987) with the F84 nucleotide substitution model (Felsenstein 1984, Kishino and Hasegawa 1989, Felsenstein and Churchill 1996). A consensus tree was assembled from the individual trees using the CONSENSE program in PHYLIP (Felsenstein 1989). Using the same set of DNA templates, all protein-coding genes were aligned with ClustalW (Thompson et al. 1994, 2002). These alignments were then concatenated to create a single alignment of 11,397 bp. A Neighbor-Joining tree was constructed using MEGA4 (Tamura et al. 2007).

RESULTS

Genome structure and composition As summarized in Table 1-1, 15,704 base pairs from Bathydorus laniger were sequenced, covering 29 genes: 12 protein-coding genes, small (rns) and large (rnl) subunit ribosomal RNA, and 15 transfer RNAs (tRNA). The protein-coding genes found were three units of cytochrome oxidase (cox], cox2, and cox3), six subunits NADH dehydrogenase (nadl, nad2, nad3, nad4, nad4L, and nad5), cytochrome b (cob), and subunits 6 and 9 of A TP synthase (atp6 and atp9). Coding regions, which include protein-coding genes, rRNA, and tRNA regions, make up 14,709 bp, or 93.7%, of the incomplete genome, while 995 base pairs are non-coding. The largest non-coding space was 262 base pairs long, spanning between cox2 and trnK (uuu). The A+ T content of B. laniger was 71.3%. 11

Coverage of the mitochondrial genome of Docosaccus maculatus was similar, with a single contiguous sequence of 17,143 base pairs, of which 1,931 were non coding-nearly double that of Bathydorus laniger (Table 1-1). The remaining 15,212 base pairs, or 88.7% of the genome, were in coding regions covering 13 protein-coding genes, the large and small subunits of ribosomal RNA, and 17 tRNAs. The protein coding genes identified were the same as those found in Bathydorus laniger, plus NADH dehydrogenase subunit 6 (nad6). Non-coding DNA was concentrated in a region between nadl and cob, with a 1,439 bp span. Areas within the non-coding region align with trnY and the 5' end of cob in two stuttering repeats before leading to the functional cob gene (Figure 1-1). The region also includes three copies of the trnY (gua); the functional trnY (gua) gene was predicted using tRNAscan-SE (Lowe and Eddy 1997) and differed by only a few base pairs from the two other copies (Table 1-2). Docosaccus maculatus has the highest ratio of non-coding to coding regions for hexactinellid mitochondrial genomes that have been sequenced to date. The A + T content for D. maculates was 70.9%.

Table 1-1. Comparison of protein-coding genes found in five species of glass sponges, and the pairwise similarities of their nucleotide and amino acid sequences.

Number ofnucleotides %Pairwise Similatitr Docosaccus Bathydorus Sympagella lphileon Aphrocallistes Start Stop Protein Nucleotide Gene maculatus lanis.er nux eanicea vastus Codon Codon @WSUM62) atp6 726 729 729 732 729 ATG TAAITA G 69.5 65.0 atp9 222 252 243 243 237 ATA TAA 81.9 84.7 cob 1,176 1,194 1,179 1,194 1,182 ATG TAGITAA 71.6 64.7 cox! 1,581 1559* 1,575 1,579 1,569 ATG TAA 78.7 65.6 cox2 735 729 729 729 741 ATG TAGITA A 74.5 72.2 cox3 783 783 783 783 784 ATG TAA 76.7 64.8 nad1 1,005 954 954 948 951 ATG TAA 73.7 68.8 nad2 1,302 1,288 1,288 1,285 1,401 ATG TAA 64.4 50.7 nad3 351 360 360 360 363 ATG TAGITA A 72.4 64.3 nad4 1125* 1197* 1383* 1,419 1,416 ATG TAA 69.4 63.1 nad4L 306 303 303 303 303 ATG TAA 70.6 61.2 nad5 1,824 1,816 1,812 1,812 1,881 ATG TAG 65.1 53.9 nad6 528 531 568 ATG TAA 54.3 31.2 ml 1,598 1,638 1,649 1,636 1,718 n.a. n.a. 73.7 n.a. ms 896 901 914 951 918 n.a. n.a. 68.3 n.a. Total coding 15,212 14,709 15,259 17,475 15,737 %coding 89 94 94 92 90 Non-coding 1,931 995 1,036 1,574 1,690 Total scguenced 17,143 15,704 16,295 19,049 17,427 *The sequence was incomplete, with some nucleotides missing from either the 5' end (cox1) or 3' end (nad4). 12

Ina d1 ,L2111Nit-----;I YI cob 1------liYI cob 1------liYI cob

Table 1-2. Variations in the trnY (gua) repeats found in the otherwise non-coding region between nadl and cob of Docosaccus maculatus. All copies were 65 bp long. The first copy was at the 5' end of the repetitive region, the third copy at the farthest 3' end; the bold, second sequence is used as a reference sequence since it was predicted to be the functional trn Y gene by tRNAscan-SE (Lowe and Eddy 1997).

Nucleotide Posit ion 29 30 32 33 48 61 First copy A ---- A G Second copy G G c G T G A Third copy T A T A c - G

Protein-coding genes All protein-coding genes were within 5% of lengths reported for other hexactinellid species with the exception of atp9, a small gene (approximately 240 base pairs) which was 7% smaller than average in D. maculatus and 5.3% larger than average in B. laniger. Pairwise percent identity ranged from 54% to 81% among nucleotide sequences ofhexactinellid species (Table 1-1). The genetic code in the two new mitochondrial genomes was consistent with the invertebrate mitochondrial code (NCBI translation table 5), as previously noted (Haen et al. 2007). Most genes used A TG as a start codon, but the start codon for atp9 was ATA for both species. Stop codons were either TAA or TAG.

Frameshifting insertions A one-nucleotide insertion, causing a shift in the reading frame of coding genes, occurred in coxl of Bathydorus laniger (somewhere between amino acid positions 70 and 13

90) and in nad2 (amino acid position 63) in Docosaccus maculatus. Each insertion was preceded by the UGG codon for tryptophan. The UGG codon was only present in coding regions before these insertions; other tryptophan residues in the genomes of Bathydorus laniger and Docosaccus maculatus were the more common UGA codon.

Gene arrangement All genes were arranged on the same DNA strand, coding in the same direction, for both species. For Bathydorus laniger and Docosaccus maculatus, a long region of synteny exists from atp6 through cox3, nad2, nad5, trn(F, C), nadl, trn(L, L N, Y), and cob; however, transfer RNA genes were highly variable both in terms of placement and presence or absence within the mitochondrial genomes, even within the region of synteny. Transfer RNA genes for trnL (uaa), trnl (gau), and trnY (gua) cluster together in the genomes of many demosponges from various clades G 1 through G4 (Wang and Lavrov 2008), and tRNA (L, I, N, and Y) are found in three hexactinellids (Sympagella nux, Bathydorus laniger, and Docosaccus maculatus). However, trnl (gau) and trnN(guu) have shifted away from that cluster in Iphiteon panicea and trnL (uaa) is missing completely from the mitochondrial genome of Aphrocallistes vastus (Figure 1-2).

Figure 1-2. Gene order in the mitochondrial genomes of five species of glass sponges. Green- protein-coding genes, red- ribosomal RNA genes, tan- tRNA, with each gene identified by the single-letter IUPAC code of the amino acid for which it codes. Multiple serine and leucine residues are differentiated as S1:trnS(uga), S2:trnS(ucu), Lt:trnL(uag), L 2:trnL(uaa), L 3 :trnL(cag). Non-coding regions are not displayed; figure is not to scale. Data for A. vastus from Rosengarten et al. (2008), data for I. panicea and S. nux from Haen et al. (2007).

Phylogenetic analysis Neighbor-Joining trees constructed from a consensus of individual gene trees had the same topology as a single tree from concatenated sequences but had lower bootstrap values (Figures 1-3 and 1-4). Taxa within the class Demospongiae clustered together, 14 including Oscarella carmela. All hexactinellids cluster together in the phylogeny, so the class appears monophyletic with present sampling.

581 Sympagella nux I 58 Bathydorus /aniger lphiteon panicea 100 I 33 I Docosaccus macu/atus

100 Aphrocallistes vastu

100 1 Xestospongia muta I - Ephydatia muelleri 75 Oscarella carmela Monosiga brevicolis

Figure 1-3. Evolutionary relationships of 9 taxa based on consensus of 12 consensus trees from protein-coding genes. Evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei 1987). The consensus tree was assembled from consensus trees of each of 12 genes: atp6, atp9, cob, coxl, cox2, cox3, nadl, nad2, nad3, nad4, nad4L, and nad5, and the percentage of individual gene trees in which the associated taxa clustered together are shown next to the branches. Each consensus tree was inferred from 10,000 bootstrap replicates (Felsenstein 1985). Evolutionary distances were computed using the F84 substitution model (Felsenstein 1984). All positions containing gaps and missing data were eliminated from the dataset. Phylogenetic analyses were conducted in SEQBOOT, DNADIST, NEIGHBOR, and CONSENSE programs within the Phylip package (Felsenstein 1989). 15

~,------Sympagella nux

100 Bathydorus laniger ,---- .------lphiteon panicea 100 ~ Docosaccus maculatus '------Aphrocal/istes vastus

100 ,------Xestospongia mula .------1 '------il '------Ephydatia muelleri 100 I.______Oscarella carmela '------Monosiga brevicolis

0.05 Figure 1-4. Evolutionary relationships of 9 taxa based on a concatenation of 12 protein-coding genes; there were a total of 10,704 positions in the final data set. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei 1987). The bootstrap consensus tree inferred from 10,000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) are shown next to the branches (Felsenstein 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). Phylogenetic analyses were conducted in MEGA4 {Tamura et al. 2007).

DISCUSSION The mitochondrial genomes of Bathydorus laniger and Docosaccus maculatus, two new species of abyssal glass sponges, bring the total number of partial genomes sequenced within Class Hexactinellida up to five. Although the region between nad4 and cox] could not be sequenced, the rest of the genomes provide over 33,000 base pairs of coverage and reveal several structural features of the two genomes.

Genome structure and composition The compact structure of the genomes, with no introns and very little non-coding space, is typical of other hexactinellid genomes that have been published (Haen et al. 2007; Rosengarten et al. 2008; Lavrov 2011), although exceptions elsewhere in Porifera exist, such as Oscarella carmela (Wang and Lavrov 2007). While non-coding regions have been found in hexactinellids, especially in Aphrocallistes vastus (10% of the genome) and Iphiteon panicea (8% of the genome), those regions do not have homology 16 to any known sequences in GenBank. The stuttering region in the mitochondrial genome of Docosaccus maculatus, on the other hand, shows homology with trnY and with the beginning of cob, although many mutations occurred in the cob areas and appear to render them non-functional. Repetitive regions have been found in demosponges (Wang and Lavrov 2007, Luki6-Bilela et al. 2008), but have not been observed in hexactinellid species, and stuttering repeats of protein-coding genes have not been observed in any other sponge genomes; the feature is so far unique to Docosaccus maculatus. The A+ T content of B. laniger was 71.3% and 70.9% for D. maculatus. These values are similar to the three other hexactinellid mitochondrial genomes that have been sequenced: Sympagella nux (70.4%), Iphiteon panicea (65.4%; Haen et al. 2007), and Aphrocallistes vastus (70.6%; Rosengarten et al. 2008).

Testing for linearity of the genome The difficulty in obtaining PCR products and sequencing results from regions of sponge DNA is well known (Lavrov 2011, Sperling and Nichols, personal communication); however, the inability to sequence the region between nad4 and cox] in both genomes coupled with previous inability to sequence that same region in Sympagella nux and Iphiteon panicea (Haen et al. 2007) brings up interesting questions about the structure of that region. I considered two hypotheses: 1) the mitochondrial genomes of these sponges are linear (Voigt et al. 2008), and PCR reactions do not work across this region because the two genes are not contiguous on a circular strand of DNA, or 2) the primary or secondary structure within this region makes PCR extremely difficult. Pulsed-field gel electrophoresis (PFGE) is standard for testing the linearity of mitochondrial genomes, and has been successful for observing the linear genomes of yeast, bacterial, and plant groups (Bendich 1993; Jacobs et al. 1996); however, PFGE requires high-quality DNA while the DNA from deep-sea species is notoriously difficult to maintain at high quality while being transported from abyssal depths to the surface. In a preliminary experiment, I tested for linearity by self-circularizing extracted DNA with T4 ligase and then attempting long PCR across the nad4-coxl region. Circularized mtDNA should be amplifiable across this gap if full-length linear mtDNA were present 17

and assuming that the termini of the molecule are 5' of cox] and 3' of nad4. The results were inconclusive: reactions did not work whether the DNA was circularized or not. However, I lacked controls for the circularizing ligation, and I could not establish whether undegraded, full-length mtDNA was present. More exhaustive experiments are needed to test this hypothesis more definitively. Stable transitions from circular to linear mapping mitochondrial DNA have occurred within closely related taxa in yeasts, algae, and fungi (Burger et al. 2003), and linear genomes have already been observed in cnidarians, lending support to this hypothesis. With the successful sequencing of Aphrocallistes vastus, the genome of at least one hexactinellid is circular, so it is likely that the genomes of other hexactinellids are circular as well. It is possible that repetitive regions, such as those discovered here, confound PCR reactions. For example, one combination of primers resulted in successful long PCR for Docosaccus maculatus from nad4 through cox] to rns. However, when sequenced, this region began 5' of coxl- not in the nad4 gene where the primer was designed to anneal. The sequenced area does not match to any other region of sponge DNA in GenBank. With the presence of cob stutters elsewhere in the mitochondrial genome of Docosaccus maculatus, it is possible that one or more repeating regions that are similar to nad4 caused mispriming in this region. To avoid this possibility, another long PCR was attempted starting from the cob region of Bathydorus laniger and the nad6 region of Docosaccus maculatus (both genes that are immediately 5' of nad4). The reaction was successful for Docosaccus maculatus, but the resulting gel showed an apparent ladder ofPCR product (Figure 1-5). Ladderized PCR products could result from multiple priming sites as would be expected from a repetitive region. I hypothesize repetitive elements exist between nad4 and cox] that hinder attempts at amplifying and sequencing the region. 18

Figure 1-5. 1.2% agarose gel prepared with sodium boric acid, run at 180V for 18 minutes. The first well contains the PCR product from a long PCR between nad6 and rns on DNA extracted from Docosaccus maculatus. The second well is a negative control, and the third is the DNA size ladder.

Protein-coding genes Most protein-coding genes typically found in mitochondrial genomes were found in these two species; a notable exception is atp8. To date, the gene has been found in all demosponges and other metazoans sampled (Wang and Lavrov 2008), but has not been found in the genomes of any hexactinellids. It has not been found in the complete genome of A. vastus, and unless it is found in the region between nad4 and cox] for the other four species, I hypothesize that it has moved to the nuclear genome in hexactinellids. Although nad6 was found in Docosaccus maculatus, it was not found in Bathydorus laniger. This gene was also not found in the sequenced region of Sympagella nux mtDNA, the closest relative to B. laniger. The region between nad4 and cox] could potentially include nad6 in these species, so I cannot conclude that nad6 is missing from their mitochondrial genomes.

Translational frameshifts Shifts occurred in the reading frames of two protein-coding genes, coinciding with the rare occurrence of the codon UGG (tryptophan). A+ 1 frameshift associated with the rare UGG codon was observed in the cox] gene of Bathydorus laniger, nad2 of Docosaccus maculatus, cox3 and nad6 of Aphrocallistes vastus (Rosengarten et al. 2008), cox] and nad2 of lphiteon panicea, and nad2 of Sympagella nux (Haen et al. 2007). Frameshifts are more common in the -1 direction (Farabaugh 1996), but+ 1 frameshifts have been found in bacteria, yeast, and metazoans (Sakai et al. 1991; Farabaugh 1996, 2000). Among metazoans, mitochondrial DNA frameshifts are known 19

in several species of birds and a turtle (Mindell et al. 1998), oyster (Milbury and Gaffney 2005), and ants (Beckenbach et al. 2005). In all five hexactinellids, the UGG (tryptophan) codon consistently preceded the + 1 frameshift, as first suggested by Haen et al. (2007) and confirmed by Rosengarten et al. (2008). One of two outcomes must result from this frameshift; the first is that the impacted gene could be a nonfunctional pseudogene and a nuclear counterpart fills its functional role (Rosengarten et al. 2008, Erpenbeck et al. 201 0). The second is that editing during transcription or some mechanism of rescue during translation must restore the proper peptide sequence (Rosengarten et al. 2008). Regions following the frameshift are continuous open reading frames that align well with functional proteins, indicating that with rescue, they might still code for coxl and nad2. A pseudogene, which does not code for a functional protein, would be expected to have more than the single insertion, so the first hypothesis is not supported by the data from hexactinellid frameshifts. The rarity of the UGG codon and its corresponding tRNA supports a mechanism proposed for programmed frameshifting (Farabaugh 1996) in which the ribosome pauses at the insertion site while the coded (but missense-creating) tRNA is sought. This pause allows enough time for a kinetically unfavorable, but more common, codon to complement the "proper" codon by skipping the inserted nucleotide (Farabaugh 1996). Rosengarten et al. (2008) notes that translational frameshifts may reduce translational efficiency, which might be selected against if there were not some other adaptive value. Farabaugh (1996, 2000) suggests that value could be as a regulatory mechanism. The presence of+ 1 frameshifts in the mitochondrial genomes of all hexactinellids sequenced to date suggests that these frameshifts are not necessarily maladaptive, as they are gained and lost frequently in the evolutionary history of the five species. Further experiments will be necessary to establish the actual consequence and potential function of+ 1 frameshifts (Farabaugh 2000).

Gene arrangement Certain areas of the genome remain relatively stable while others are highly dynamic. The major region of synteny between atp6 and cob observed by Rosengarten et al. (2008) is still maintained with the addition of these two new genomes; however, the 20 large, stable area of synteny is flanked by regions that have undergone several transpositions. The region surrounding rns changes with each species. In some species, rns and rnl are adjacent to each other, while in Docosaccus maculatus the ribosomal RNA genes are separated by cox2 (Figure 1-2). Also in that region, atp9 and cox] are very mobile and appear in different arrangements in many of the hexactinellid mitochondrial genomes. These results show that gene arrangement can be highly variable, but does not appear to be random. Many of the major transpositions and gene losses are supported by the phylogenetic placement of each species, as summarized in the cladogram in Figure 1-6.

Docosaccus maculatus atp9 nad2 trnW shift (pos 63) trnL (uag) rns cox2 ml Iphiteon panicea trnl trnN nad2 cox/ (pos 335) (pos 225)

atp8

Sympagella nux m ,, rnl nad2 (pos 63)

cox/ Bathydorus laniger

Aphrocallistes vastus cox3 nad6 (pos 140) (pos 58)

Figure 1-6. Cladogram representing some major changes in mitochondrial gene arrangement with phylogenetic context. Red bars indicate losses of genes or frameshifts (although the loss of nad6 may be a shift into the unsequenced region between nad4 and coxl), blue bars indicate gene shifts, and green bars indicate + l frameshifts caused by tryptophan (UGG) codons.

Phylogenetic analysis Tree topologies from a consensus of individual protein-coding gene trees and a tree inferred from the concatenation of all protein-coding genes were the same. The topology does not, however, match topologies inferred from morphological evidence (Hooper and Van Soest 2002). Jphiteon panicea is part of the family Dactylocalycidae, 21

which, along with Aphrocallistes vastus, falls under the order Hexasterophora (Hooper and Van Soest 2002). However, in the trees presented here, lphiteon panicea clusters more closely with Docosaccus maculatus, a member of the order Lyssacinosida. The topology of the present study is similar to that found using 18S ribosomal regions, and so is now supported by trees from multiple data sets (Dohrmann et al. 2009). An investigation of museum specimens and morphological characters of I. panicea should be used to determine whether the species belongs elsewhere in sponge systematics, and sequences from more specimens from the family Dactylocalycidae should be evaluated to determine whether I. panicea or the entire family should be re-evaluated. Demosponges clustered together along with Oscarella carmela, although the homoscleromorph was the most distantly related of the three species evaluated. Oscarella carmela shares several features with bilaterians that other sponges do not, such as striated cilial rootlets, type IV collagen, and possibly a true epithelium (Wang and Lavrov 2007). The GO clade within demosponges, the homoscleromorphs, has appeared to be monophyletic and possibly distinct from other demosponge clades; its location in the trees of this study supports these other analyses (Wang and Lavrov 2007, 2008; Lavrov et al. 2008, Lavrov 2011 ).

CONCLUSIONS The mitochondrial genomes ofhexactinellids are structurally dynamic and diverse. Gene order is stable in some areas while highly variable in others, and genes coding for transfer RNAs move more frequently than protein-coding genes. Unique structural features exist, such as frameshifts caused by + 1 insertions and the non-coding region in Docosaccus maculatus that contains stuttering repeats of coding sequences. Future studies will be necessary to evaluate the mechanisms behind these unique features. Finally, phylogenies inferred from mitochondrial gene sequence data suggest that the placement of Iphiteon panicea be re-evaluated. 22

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CHAPTER2

BATHYDORUS LANIGER AND DOCOSACCUS MACULATUS (LYSSACINOSIDA; HEXACTINELLIDA): TWO NEW SPECIES OF GLASS SPONGES FROM THE ABYSSAL EASTERN NORTH PACIFIC OCEAN

AMANDA S. KAHN1'4, JONATHAN B. GELLER1, HENRY M. REISWIG2, & KENNETH L. SMITH, JR. 3 1Moss Landing Marine Laboratories, 8272 Moss Landing Road, Moss Landing, CA 95039, USA. E-mail: [email protected]. [email protected] 2 Department ofBiology, University of Victoria and Natural History Section, Royal British Columbia Museum, Victoria, British Columbia, V8W 3N5, Canada. E-mail: hmreiswig@shaw. ca 3Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA. E-mail: [email protected]. 4 Corresponding author Formatted for the journal Zootaxa

ABSTRACT Two new species of glass sponges were discovered from the abyssal plain 200 km west ofthe coast of California (Station M). The sponges have similar gross morphology-a unique plate-like form with basalia stilting the body above soft abyssal sediments. Bathydorus laniger sp. n. differs from its congeners by the presence of dermal and atrial stauractins; it is also supported by smooth hypodermal pentactins and hypoatrial hexactins. Microscleres include oxyhexasters and oxyhemihexasters. Docosaccus maculatus sp. n. contains large hexactins (> 1 em), characteristic of the genus. Megascleres include dermal hexactins, atrial pentactins, and choanosomal hexactins and diactins. Microscleres include oxy-tipped hemihexasters and floricomes. Descriptions of 27 these new species are timely as studies will soon emerge establishing sequences of their mitochondrial genomes and revealing population-level changes using time-series data from Station M.

INTRODUCTION Gross body morphology of sponges, which includes tubular, vase-like, and encrusting shapes, can vary depending on surrounding current, water flow, and oceanographic conditions (Palumbi 1984, 1986; Bell and Barnes 2000). Thus, while gross morphology is not important in assigning taxonomic position, it does reflect an ecological response to localized conditions. Taxonomic categorization of sponges uses primarily hard skeletal components, the spicules, for its organization. Two new species of glass sponges were found from the same area in the eastern North Pacific; their spicule composition places them in two separate families within Order Lyssacinosida (Reiswig 2002), yet both share a similar plate-like morphology. Glass sponges occur in many shapes, including stalked, encrusting, massive, tube like, vase-like, and plate-like, or flat. Although reference to a plate-like morphology is made in Systema Porifera (Hooper & Van Soest 2002), the current taxonomic reference for higher-level poriferan systematics, no specific genera are mentioned as having that morphology. Inquiries with researchers at the video analysis laboratory of the Monterey Bay Aquarium Research Institute yielded no recollection of plate-like sponges in any of their hundreds of hours of video. The discovery of two species with plate-like morphology, yet of different families, in the same area prompts questions regarding the ecological significance of their morphology. The two new species were found at Station M, a long-term study site in the abyssal northeast Pacific (Figure 2-1; Smith and Druffel 1998). The particular conditions at Station M that may have prompted plate-like morphology cannot be determined with present data; however, several parameters have been measured at this site and are summarized below, for comparison with conditions of future discoveries of plate-like sponges. Similarities between the sites will help form hypotheses regarding the cause of a flat body shape. 28

Station M is characterized by low topographic relief (less than 100 m over a 1,600-km2 area), silty-clay sediments, and current speeds of2.19±1.76 em s·1 at 2.5 m above the seafloor (Beaulieu and Baldwin 1998). Particulate organic carbon (POC), the food source for deep-sea communities, rains from the seasonally productive California Current, a strong upwelling system that varies greatly in primary production (Smith et al. 2006). Carbon and other nutrient concentrations, sediment community oxygen conswnption rates, megafauna! activity, and animal abundance have been monitored from 1989 until present with multiple measurements per year. A series of camera sled transects recorded variations in multiple species of plate sponges (91.2 ± 12.4 sponges/ha) throughout the time-series (Kahn et al. in prep), with plate-like sponges more abundant following times of higher POC flux to the seafloor.

Figure 2-1. Collection location of two new species of glass sponges with a unique plate-like morphology: Station M ( 4,100 m depth, 34°50'N, 123°0'W), a long-term abyssal study site in the northeast Pacific.

MATERIALAND METHODS Twelve specimens were collected from Station M (4,100 m depth) in 2007 by remotely operated vehicle (ROV Tiburon). Holotypes and other specimens for both species were found during cruises to Station M in June and November 2007. Before 29

2007, specimens were obtained by otter trawls during various cruises to the long-term study site. Sponges collected by ROY were photographed in situ using high-definition cameras. A manipulator arm gently transferred them into a biological collection box with minimal sediment disturbance. Little temperature change (<5°C) occurred as sponges were brought to the surface, and once at the surface specimens were immediately subsampled for DNA analysis. Duplicate DNA samples were taken; one deep-frozen in liquid nitrogen, then transferred to -80°C and the other simply stored in 90% ethanol. The rest of the sponges were preserved in 10% buffered formalin. Additional sponge samples were collected by an otter trawl attached to a towed camera sled (Lauerman et al. 1996). Samples from the tows were bulk preserved in 10% formalin and later sorted by the Benthic Invertebrate Collections at Scripps Institution of Oceanography. Sponges obtained this way were crushed and broken into pieces, but enough material existed that they could be matched with the holotypes. Permanent spicule preparations were made by digesting tissue fragments from the dermal, choanosomal, and atrial regions in hot nitric acid for at least 1.5 hours. Once all organic material was cleaned off, spicules were concentrated and isolated for later light microscopy or scanning electron microscopy (SEM) applications. For light microscopy, cleaned spicules were concentrated on 0.2 )liD pore-size nitrocellulose filters. Filters were placed on a glass microscope slide, rinsed in xylene three times, then rinsed once more in ethanol. Finally, a drop of Canada balsam and a cover slip permanently sealed the filter. For SEM work, spicules were either concentrated on polycarbonate membrane filters or hand-picked and transferred individually onto SEM pegs. Pegs were sputter-coated with gold-palladium and viewed in a Hitachi S-3500 scanning electron microscope at the University of Victoria and at Moss Landing Marine Laboratories. Holotype and paratype specimens will be stored in the benthic invertebrate zoology collection at Scripps Institution of Oceanography. Abbreviations: Scanning electron microscopy (SEM) light microscopy (LM), Scripps Institution of Oceanography Invertebrate Zoology Collection (SIO-IZ), Monterey Bay Aquarium Research Institute (MBARI), and Moss Landing Marine Laboratories 30

(MLML). Terminology follows that ofTabachnick and Reiswig (2002) and Boury Esnault and Riitzler (1997).

RESULTS Class Hexactinellida Schmidt, 1870 Subclass Hexasterophora Schulze, 1886 Order Lyssacinosida Zittel, 1877 Family Rossellidae Schulze, 1885 (Gray 1872) Subfamily Rossellinae Schulze, 1885 Genus Bathydorus Schulze, 1886 Type species: Bathydorus fimbriatus Schulze, 1886 (by subsequent designation; Koltun, 1967). Genus diagnosis. Rossellinae with tubular, saccular, or plate-like gross morphology. Basiphytous or lophophytous, thin-walled. Dermalia are combinations of spicules from hexactins to diactins. Regular pentactins make up a hypodermal layer. Choanosomal skeleton composed of diactins, sometimes with hexactins. Atrialia are hexactins or stauractins. Microscleres are combinations of oxyoidal hexasters, hemihexasters, and hexactins; lacking pappocomes (From Tabachnick 2002b: 1463, emended). Remarks. The diagnosis of the genus is emended to include the plate-like gross morphology, lophophytous method of attachment, and atrial stauractins that occur in the new species. Bathydorus laniger sp. n. (Figures 2-2 through 2-4, Table 2-1)

MATERIAL EXAMINED. Holotype: Stored at MBARI, coli. A. S. Kahn using MBARI ROV Tiburon, dive T1094 from R/V Western Flyer, 3,950 m depth, Station M (34°50'N, 123°0'W), 05 June 2007. Paratypes: MBARI Sponges 1a-lb, 2a-2d coli. H. Ruhl using MBARI ROV Tiburon, September 2007 from R/V Western Flyer, 4,000 m depth, Station M, 21-23 September 2007; SIO-IZ P1463, coli. K. L. Smith using otter trawl, PULSE 46, depth ~4, 100 m, Station M, February 2005.

DIAGNOSIS. Bathydorus with dermal and atrial layer of stauractins; hypodermal pentactins smooth, microscleres usually only oxyhexasters and oxyhemihexasters, small 31 oxyhexactins also rarely found. Body shape plate-like with dermal surface facing downward, toward the seafloor. A fringe of marginal prostalia protrudes from the perimeter and solitary pleural prostalia project from the dermal surface, inserting into the substrate and providing anchorage. No major oscula present, but usually one small hole in center of dermal surface may represent a residual osculum; color white.

DESCRJPTION. The holotype and other paratypes stored at MBARI were collected whole by ROV. The paratype stored at SIO-IZ was assembled from fragments from otter trawls. Holotype (Figure 2-2) 38 em diameter at longest axis, 1-3 mm thick. The atrial surface is smooth and faces up away from seafloor, while the dermal surface contains long (over 5 em long), solitary prostal diactins that project down into the sediments and anchor the sponge. Marginal prostals project from the perimeter forming a fringe around the sponge, and are angled slightly toward the sediments. Neither surface has recognizable ostial or oscular apertures, but spacing between nodes in the stauractin framework is 78.8 ± 9.4 !J.m (mean± SD). A large hole perforates the center, leaving a concavity in the atrial surface. Live and preserved specimens are white, with a crunchy but pliable texture.

Figure 2-2. Bathydorus laniger sp. n. holotype, whole body images. A. In situ photograph of whole organism taken by ROV Tiburon. Atrial surface with marginal prostalia visible. Credit: MBARI. B. Dermal surface, with pleural prostalia (P) seen protruding as long, single diactins. Scale bar: 10 em.

LYSSACINE FRAMEWORK. Moving from the lower dermal to the upper atrial surface, the outermost layer of the dermal surface is a single layer of stauractins, networked together but not fused. Below.the stauractin layer are large, smooth, hypod rmal pentactins arranged in a semi-regular arrangement, with proximal rays 32

pointing toward the choanosome and tangential rays nearly aligning with the distal stauractin network. The choanosomallayer is primarily comprised oflong diactins but also contains scattered oxyhemihexasters. A layer ofhypoatrial hexactins is irregularly arranged distal to the choanosome, and a final layer of networked stauractins finishes off the atrial surface (summarized in Figure 2-3).

,("'' J I ·~,tl ""- '1 ~ ''lo ,~~~'· ...., ,/ ,,,, /' ' / ""<'7 / '>t,./ I ' ../ /tf ' ,/ '",,,, ,(f" '1:;,1 ~~,;; / ..{' ·~.~\,. II 1'~- A ,_' ~A'/. ~ ,t ;:;r 1rt /' ;z, # ~-~~ ~y ./ ~' -~, ,11" \ . ..c'R/ ~,;A ? -~ ''. ,~~ ·<~, ''?<' -", -)z~.... / ' "'t;,/1 jr. ,(1 / '<\,, ''}/( /' ,.r"' -.,,., .}~ · '')'\'' II/' 7 / (/'~ ' 'v// .J~'"''" I , I / "- />'~~"'-. I' """ ~~- \l --\v( -V- 1 --\~ ~ I 'L I' -;r- __.y 1\ ~f' /\ -""' 7\ ------==

Figure 2-3. Bathydorus laniger sp. n. from Station M, California, USA; scale diagram of spicule arrangement. The atrial surface is at the top of the image while the dermal surface is at the bottom; prostal diactins are omitted. Scale bar: 200 Jtm. 33

The channel system could not be determined, but the dermal surface appears to have regularly spaced openings among the stauractin network (80J..Lm diameter) while the atrial surface has a fine mesh with no distinctive oscula (Figure 2-3).

SPICULES. Spicule forms are shown in Figures 2-3 and 2-4 and dimensions are provided in Table 2-1. Megascleres include stauractins, pentactins, diactins, and hexactins. Microscleres range from oxyhexactins to oxyhexasters and oxyhemihexasters. The outermost dermal layer is a network of rough stauractine dermalia (Figure 2-4a) with a layer of large, smooth hypodermal pentactins (Figure 2-4c) immediately proximal to it. Oxyhexasters and oxyhemihexasters with one to three secondary rays branching from each primary ray (Figure 2-4e) are scattered among the longer proximal rays of the pentactins. Long choanosomal diactins in the center provide internal structure along with the proximal rays of the pentactins. A swelling halfway along the length of the diactins contains the axial cross and the four vestigial axial filaments that would make up a hexactine spicule; the tips are slightly rough (Figure 2-4d). The outem1ost atrial surface also begins with a layer of rough stauractine atrialia, followed immediately by a proximal, hypoatriallayer of rough hexactins with all rays approximately the same length (Figure 2-4b). Diactine prostalia project as both marginalia and pleuralia to provide anchorage in the sediments, acting as functional basalia. Long diactins project tangentially from the atrial surface or margin. Thin protein strands spiral around the proximal ends of the prostalia, but they diminish as distance from the main body increases. No central swellings or tubercles are observed along the length of the diactins. The tips of the prostalia were smooth and each tapered to a point, but did so in discrete, incremental reductions in width. No structural differences were observed between diactins comprising pleural prostalia versus marginal prostalia, except that marginal prostalia did not curl as much as pleural prostalia. 34

Figure 2-4. Bathydorus laniger sp. n., spicule images from SEM. A. Dermal stauractin. B. Atrial hexactin. C. Hypodermal pentactin. D. Tip of choanosomal diactin. E. Oxyhexaster, oxyhemihexasters, and oxyhexactin. Scale bar: 200 J.tm.

ETYMOLOGY. The species name, laniger, refers to the fringe of spicules along the perimeter of the sponge, giving it a "hairy" appearance.

REMARKS. To our knowledge, this species has thus far not been found elsewhere in the world. The arrangement of spicules clearly identifies this species as a member of the genus Bathydorus; however, the plate-like gross morphology along with the layer of stauractine atrialia differentiates it from others within the genus. The new species differs from the six known species of Bathydorus (and four sub species) by a variety of characters. The unique gross morphology does lend itself as a character for species identification, but is not reliable because sponges can change morphology based on surrounding conditions (Palumbi 1984). Bathydorus laniger has a layer of atrial stauractins that is only otherwise found in Bathydorus uncifer Schulze, 1899; all other members of the genus have atrial hexactins. Bathydorus uncifer has pentactins in the atrial surface along with the stauractins, plus it is only found in the 35

equatorial Pacific near the Galapagos Islands (Schulze 1899). Bathydorus uncifer also contains hypodermal and hypoatrial stauractins, both of which are missing in B. laniger. While atrial pentactins easily differentiate B. laniger from the rest of its congeners, other differences also exist. Bathydorus laevis and its subspecies have hexactins with varying degrees of roughness (Schulze 1886, 1902; Wilson 1904, Koltun 1967), while all hexactins in B. laniger are uniformly rugose. The hexactins of B. spinosissimus resemble pinules, with large spines on the proximal ray directed toward the tip that progressively increase in size down the length of the ray (Lendenfeld 1915), which is very different from the round, symmetric rays of B. laniger. Like B. laniger, the dermal surface of Bathydorus echinus Koltun, 1967 is composed of dermal stauractins, but contains pentactins and hexactins as well (Koltun 1967). Similarly, B. servatus Topsent, 1928 has dermal stauractins as well as diactins (Topsent 1928). Bathydorus fimbriatus Schulze, 1886 has only stauractins in its dermal surface, but the hexactins in its atrial surface are not found in B. laniger (Tabachnick 2002b). In view of these differences, we conclude that B. laniger is a new species, bringing the total number of Bathydorus species to seven.

Table 2-1. Spicule dimensions of Bathydorus laniger sp. n., from Station M, California, USA (dimensions in J.lm, except for the choanosomal and prostal diactin lengths, which are in mm).

~arameter mean SD range number Megascleres Hypoatrial hexactin ray length 83.8 13.7 63.8- 111.4 22 ray width 6.4 1.3 3.6-8.9 25 Hypodermal pentactin tangential ray length 304.1 46.8 173.7- 383.0 50 tangential ray width 18.9 4.3 6.2- 26.3 50 proximal ray length 449.5 74.2 335.2- 792.3 50 proximal ray width 22.9 5.4 10.0- 36.2 50 Dermal and atrial stauractin ray length 78.8 9.4 53.2-98.1 50 ray width 5.5 0.9 3.7- 8.0 50 Choanosomal diactin length (mm) 20.9 6.2 13-34 20 Prostal diactin length (mm) 130.4 39.8 71 - 210 20 Microscleres Oxyhemihexaster diameter 120.8 14.8 82.1-151.2 50 Primary ray length 9.2 1.9 4.3- 13.2 50 secondary ra~ length 56.5 8.9 33.6-77.5 50 36

Family Euplectellidae Gray, 1867 Subfamily Euplectellinae Gray, 1867 Genus Docosaccus Topsent, 1910 Type species: Docosaccus ancoratus Topsent, 1910 Genus diagnosis. Lophophytous, body sac-like or plate-like, with thin walls fixed by several tufts of anchor-like basalia or bundles of diactins surrounding a giant hexactin. Choanosomal spicules are diactins, rarely hexactins and their derivatives. Largest choanosomal spicules are hexactins with long tangential rays or rays of varying size. Dermalia are hexactins, atrialia are hexactins or pentactins. Microscleres are hexactins, I hemihexasters, hexasters, floricomes, and probably discohexasters (Tabachnick 2002a: 1395-1396, emended). Remarks. The diagnosis of the genus is emended to include the plate-like gross morphology, anchoring spicules, and atrial pentactins that occur in the new species. Docosaccus maculatus sp. n. (Figures 2-5 through 2-8, Table 2-2)

MATERIAL EXAMINED. Holotype: MBARI "Sponge 2", coll. A. S. Kahn using MBARI ROV Tiburon, dive Tl094 from R/V Western Flyer, 3,950 m depth, Station M (34°50'N, 123°0'W), 05 June 2007. Paratypes: MBARI "Sponge 3", coll. A. S. Kahn using MBARI ROV Tiburon, dive 1094 from R/V Western Flyer, 4,000 m depth, Station M, 06 June 2007; MBARI "3a", coll. H. Ruhl using MBARI ROV Tiburon, September 2007 from R/V Western Flyer, 4,000 m depth, Station M.

DIAGNOSIS. Docosaccus with dermal layer of rough hexactins and atrial layer of rough pentactins; microscleres oxyhexasters, oxyhemihexasters, and floricomes. Body shape flat, plate-like, with smooth margins (no marginal prostalia). Lophophytous mode of attachment, each anchoring spicule tuft with bundles of diactins surrounding a giant hexactin. Dermal surface facing downward, toward the seafloor. Parietal oscula regularly scattered across the surface; color translucent white.

DESCRIPTION. The holotype and paratype were collected whole by ROY. Holotype (Figure 2-5) 13.8 em diameter at longest axis, 1-3 mm thick. The atrial surface is smooth, facing up away from seafloor, while the dermal surface shows several tufts of anchoring basalia, consisting of diactins surrounding a single large hexactin. Both 37 surfaces have regular ostial or oscular apertures. Also scattered across the body are larger holes, parietal oscula, 2 to 5 mm in diameter, which perforate the entire body and appear as dark spots in photographs. Both live and preserved specimens are translucent white, and in photographs appear to have white and black spots (Figure 2-5). The black spots appear where parietal oscula perforate the sponge body while white spots appear where anchoring spicule tufts project from the dermal surface. Spicules are loosely arranged, resulting in a flexible, delicate texture that tears easily.

Figure 2-5. Docosaccus maculatus sp. n., whole body images of holotype. A. In situ photograph taken by ROV Tiburon. Atrial surface with black and white spots visible. Black spots are caused by shadows in holes that perforate the body (parietal oscula), white spots occur where anchoring spicule tufts are visible through the body. Credit: MBARI. B. Dermal surface, showing small papillae consisting of anchoring spicule bundles. cale bar: 5 em.

LYSSAC INE FRAMEWORK. Framework is loose, resulting in a flexible but delicate structure that is easily broken. Moving from the dermal to the atrial surface, the outermost layer of the dem1al surface is a single layer of sword-shaped hexactins with tangential rays forming an unfused, lyssacine network (Figure 2-6 bottom). Proximal rays are much longer than the others, and project all the way through the choanosome to the atrial surface. Floricomes perch atop the distal ray of the hexactins. Below the dermal layer is a mesh of long diactins, generally pointing in all directions but sometimes forming bundles or tracts. Among the diactins are large, rough hexactins with even rays. 38

Distal to the choanosome is the atrial membrane composed of pentactins arranged with an overlapping network of tangential rays (summarized in Figure 2-6 upper).

Figure 2-6. Diagram of spicule arrangement for Docosaccus maculatus sp. n. from Station M, California, USA. Scale bar: 200 J.lm.

The channel system is compressed into just two millimeters of body thickness, yet contains circuitous channels distributing water flow through the body. Openings of inhalant canals are regularly spaced across the dermal surface (about 100 to 250 J.lm diameter), and oscula are regularly spaced and visible on the atrial surface (150 to 500 39

!lm diameter). Internal channels leading from inhalant canal apertures and oscula curve laterally and presumably branch and weave through the choanosome.

SPICULES. Spicule forms are shown in Figures 2-6 through 2-8 and dimensions are provided in Table 2-3. Megascleres are hexactins of three varieties, rough atrial pentactins, and choanosomal and anchor-tuft diactins. Microscleres are oxyhexasters, oxyhemihexasters, and floricomes. The three types ofhexactins will be described as 1) giant hexactins, 2) dermal hexactins, and 3) choanosomal hexactins. Giant hexactins are smooth, with no spines along the entire length. A single giant hexactin was found in each anchoring spicule tuft; the sinuous, thickened distal ray protrudes down into the sediments along with a bundle of diactins. The distal, anchoring ray and one tangential ray running through the choanosome are of similar dimensions (21-25-30 mm, min-mean-max, n=4). Though not oriented in any regular direction, the long tangential ray overlaps with those of other giant hexactins and reinforces structure throughout the body. Two other tangential rays are smaller (4-7.2-14 mm, n=4) while the final tangential ray and the proximal ray are just reduced, rounded swellings of about 100 f!m in length. The sword-shaped dermal hexactins form a network of overlapping tangential rays, with longer proximal rays pointing into the choanosome (Figure 2-7 c). These hexactins are covered with small spines; tangential rays are covered evenly while the proximal ray has more spines near the center and fewer at the tip. The distal ray is also covered with spines, and tapers to a point like the other rays. Choanosomal hexactins are covered with spines evenly throughout the length of the six cylindrical, pointed rays (Figure 2-7b ). Rays of choanosomal hexactins are of roughly equal size, unlike those of the dermal and giant hexactins. Atrial pentactins form a network of overlapping tangential rays, with proximal rays pointing into the choanosome. Pentactins are rough with spines, but the spines are not as large as those of the dermal hexactins. Tangential rays are straight, while the proximal ray is either straight or bent slightly near the base (Figure 2-7a). Microscleres include oxyhexasters, oxyhemihexasters, and floricomes. Full oxyhexasters with all six primary rays bearing two or more secondary rays are the most common form, but oxyhemihexasters with at least one set of multiple secondary rays (1-3 40 secondary rays for each primary ray) are also present. Both oxyhexasters and oxyhemihexasters are smooth, with short primary rays and tapering secondary rays that end in pointed (oxyoidal) tips (Figures 2-7d through 2-7g). They are found scattered throughout the sponge, from just below the dermal surface to the atrial membrane. Floricomes are found in the dermal membrane perched atop the distal rays of dermal hexactins. They have 9-12 S-shaped secondary rays radiating from each short primary ray (Figure 2-8). Each secondary ray is roughened with small spines on the concave part of the distal curve, and widens to a flattened, eccentric toothed claw at the end with 4-6 teeth. Table 2-2. Spicule dimensions of Docosaccus maculatus sp. n., from Station M, California, USA (dimensions in 11m).

parameter mean SD range number Megascleres Atrial pentactin tangential ray length 326.9 42.7 241.3-401.0 16 tangential ray width 28.3 4.5 21.3-37.0 16 proximal ray length 653.2 139.0 426.6-880.0 17 proximal ray width 29.5 2.8 24.6-36.1 15 Choanosomal hexactin ray length 180.2 107.6 56.7- 596.1 34 ray width 9.2 5.2 2.8- 30.0 34 Dermal hexactin tangential ray length 340.9 68.8 195.0-466.8 27 tangential ray width 20.9 5.7 8.9- 31.6 50 proximal ray length 596.5 164.7 356.5-1134.9 49 proximal ray width 22.3 5.8 10.7- 37.3 49 distal ray length 141.9 38.9 66.2-232.8 50 distal ray width 20.1 5.1 10.4-32.4 50 Microscleres Oxyhemihexasters & oxyhexasters diameter 79.1 7.2 63.2- 93.8 50 primary ray length 7.5 1.3 4.1- 10.1 50 secondary ray length 31.9 3.8 24.3-41.7 50 Floricome diameter 79.1 7.2 63 .2-93.8 50 primary ray length 8.3 1.6 5.2- 11.9 22

secondary ray length 43.9 6.5 ° 28.4-51.8 22 41

·. ' ~ I '·I ;

Figure 2-7. Spicule images of Docosaccus maculatus sp. n. (LM). A. Atrial pentactin. B. Choanosomal hexactin. C. Dermal hexactin. Scale bar (between Figures 7a and 7b): 200 Jtm. D-G. Oxyhexasters. Scale bar (between 7d and 7f): 50 Jtm. b

Figure 2-8. Floricomes of Docosaccus maculatus sp. n. (SEM). A. Complete floricome. Scale bar: 50 Jtm. B. Close-up of floricome secondary rays. Scale bar: 10 Jtm. 42

ETYMOLOGY. The species name, maculatus, refers to the white and dark spots that appear under strong light in situ. Parietal oscula perforating the entire body wall appear as dark spots while tufts of anchoring basalia, viewed through the atrial surface, appear as white spots.

REMARKS. To our knowledge, this species has not been found elsewhere in the world. Its only congener, Docosaccus ancoratus Topsent, 1910, is found frequently but only in Antarctic waters (Barthel and Tendal 1994). The unique giant hexactin identifies the two species as closely related and belonging to the same genus, but geographic distribution and spicule composition confirm that they are different species. Docosaccus maculatus has an atrial membrane supported by pentactins while the atrial membrane of D. ancoratus is supported by hexactins. Oxyhexasters and oxyhemihexasters are much thicker in the Pacific species, appearing much more delicate in the plates from Topsent (1913). The dermal hexactins have pointed distal rays in D. maculatus, while they appear rounded into a spined swelling in the Antarctic species. Finally, the anchoring tufts are different between the two species. The diactins surrounding the giant hexactins of D. maculatus are straight and smooth with unevenly tapering tips while those of D. ancoratus are covered with spines recurved back toward the body and ends that look like grappling hooks (Topsent 1910, 1913). These differences confirm that the NE Pacific form is a new species, here designated as Docosaccus maculatus.

DISCUSSION The two n~w hexactinellid species at Station M, Bathydorus laniger and Docosaccus maculatus, increase the diversity of their respective genera in terms of geographic distribution, gross morphology, and spicule arrangement. Though the two species are clearly of different families based on their spicule composition, their gross morphology is very similar and not frequently noted for other hexactinellid species. Convergence of such a unique morphology in two different families found in the same area prompts future ecological studies. Currently, one ecological study has been done on these two species. A seventeen year time-series examined changes in population density and average size of plate 43 sponges through time and with changing POC flux to the seafloor. Results of that study will be published in a forthcoming paper (Kahn et al., in prep). Other ecological observations of these sponges also should serve to inspire future studies. Many sponges were found growing on spicule mats of dead sponges. These mats may provide enough heterogeneity to disrupt currents and enhance larval recruitment (Bett and Rice 1992), but it could also be that the sponges growing on those mats grew back from a few persistent cells of the only apparently dead sponge. Molecular analysis will help address this question. Most other members of Bathydorus, and certainly Docosaccus ancoratus, are sacciform or tubular. This morphology orients the dermal surface perpendicular to boundary layer currents. With plate sponges, however, the entire dermal surface faces down toward the sediments. Laminar flow of boundary layer currents passing through basalia would be disrupted, and the turbulent eddies that result may resuspend sediment or pore water from the sediments. This melange of horizontally advected water and pore water would be filtered through the sponge for food. If this model is correct, it means that plate sponges are able to take advantage of food sources from both pelagic and benthic sources. While the future ecological studies are intriguing, the contribution that these two species provide to their respective genera is valuable on its own. Additionally, taxonomically characterizing the species found at Station M provides greater detail to the time-series studies and provides opportunities for future ecological investigations. 44

REFERENCES

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Reiswig, H. M. (2002). Order Lyssacinosida Zittel, 1877. In: J. N. A. Hooper and R. W. M. Van Soest (Eds.). Systema Porifera: A Guide to the Classification ofSponges . Kluwer Academic/Plenum Publishers, New York. p. 1387. Schulze, F. E. (1886). Uber den Bau und das System der Hexactinelliden. Abhandlungen der Koniglichen Akademie der Wissenschaften zu Berlin (Physikalisch Mathematisch Classe), pp. 1-97. Schulze, F. E. (1899). Amerikanische Hexactinelliden, nach dem Materiale der Albatross-Expedition. (Fischer: Jena), 1-126, pls I-XIX. Schulze, F. E. (1902). An account ofthe Indian triaxonia collected by the Royal Indian Marine Survey Ship Investigator. Translated by R. von Lendenfeld. Indian Museum, Calcutta, India, 113pp. Smith, K. L. , Jr., R. J. Baldwin, H. A. Ruhl, M. Kahru, B. G. Mitchell, and R. S. Kaufmann (2006). Climate effects on food supply to depths greater than 4,000 meters in the northeast Pacific. Limnology and Oceanography, 51(1): 166-176. Smith, K. L., Jr., and E. R. M. Druffel (1998). Long time-series monitoring of an abyssal study site in the NE Pacific: an introduction. Deep-Sea Research II, 45:573-586. Tabachnick, K. R. (2002a). Family Eup1ectellidae Gray, 1867. In: Systema Porifera: A Guide to the Classification ofSpong es, J. N. A. Hooper and R. W. M. Van Soest (Eds.). Kluwer Academic/Plenum Publishers, New York. pp. 1388-1434. Tabachnick, K. R. (2002b). Family Rossellidae Schulze, 1885. In: J. N. A. Hooper and R. W. M. Van Soest (Eds.). Systema Porifera: A Guide to the Classification of Sponges. Kluwer Academic/Plenum Publishers, New York. pp. 1441-1505. Tabachnick, K. R., and H. M. Reiswig (2002). Dictionary ofHexactinellida. In: J. N. A. Hooper and R. W. M. Van Soest (Eds.). Systema Porifera: A Guide to the Classification ofSponges. Kluwer Academic/Plenum Publishers, New York. pp. 1224-1229. Topsent, E. (1910). Les Hexasterophora recueillies par la "Scotia" dans !'Antarctique. Bulletin de l'Institut Oceanographique, 166:1-18. Topsent, E. (1913). Spongiaires de !'Expedition Antarctique Nationale Ecossaise. Transactions ofthe Royal Society ofEdinburgh , 49(3, 9):579-643, 6 pls. Topsent, E. (1928). Spongiaires de 1' Atlantique et de la Mediterranee provenant des croisieres du Prince Albert ler de Monaco. Resultats des campagnes scientifiques accomplies par le Prince Albert I. Monaco, 74:1-376, pis I-XI. Wilson, H. V. (1904). Reports on an Exploration off the West Coasts ofMexico, Central and South America, and off the Galapagos Islands, in charge of Alexander Agassiz, by the U. S. Fish Commission Steamer 'Albatross' during 1891, Lieut. Commander Z. L. Tanner, U. S. S., commanding. XXX. The Sponges. Memoirs of the Museum of Comparative Zoology at Harvard College, 30(1):1-164, pls. 1-26. 46

APPENDIX A

GENBANK ACCESSION ENTRIES FOR BATHYDORUS LANIGER AND DOCOSACCUS MACULATUS 47

Locus Bathydorus_lanig 15704 bp DNA linear UNA 28-NOV-2010 DEFINITION Bathydorus laniger mitochondrion, partial genome ACCESSION urn: 1ocal:. :1290225953296.16 VERSION KEYWORDS Bathydorus, Rossellidae, mitochondrion, genome, mitochondrial SOURCE mitochondrion Bathydorus laniger (fringed plate sponge) ORGANISM Bathydorus laniger Eukaryota; Metazoa; Porifera; Hexactinellida; Hexasterophora; Hexactinosida; Rossellidae; Rossellinae; Bathydorus FEATURES Location/Qualifiers mise feature 1606 .. 1737 / note="Geneious type: ORF" rRNA 3298 .. 4935 /gene="rnl" /product="large subunit ribosomal rRNA" rRNA 2396 .. 3296 /gene="rns" /product="small subunit ribosomal RNA" CDS 10064 .. 11959 /gene="nad5 " /codon_start=1 /transl table=5 /product="NADH dehydrogenase subunit 5" / translation="MYLLMMSTPLMSFILTFMNQKRMRPNTIQTLACMIMLFAWMNSV TMFYECMMNDTHCQTKIINWMEMVHMSSKMSLHMDTLSSSMLFI I TSISLFVHVYSLQ YMNEDPHKHRFISYLSLFTFFMMILVSSNNFMLLLMGWEGVGICSYLLMNFWYTRIQA NKSGMKAMMINRMGDMALLMSTMIIMKKFGSTKFENLIHMQENIKMQFTMNSICLLLL MAAMGKSSLMGLHIWLPDAMEGPTPISALMHAATMVTAGIFLLIRSSSLLEESKMSLM MTAWIGAMTAFFAATTGLSQNDMKRIMAYSTCSQLGYMALAIGISKYNIGLLLLLILY SLKDYYSMGAGIAI HTMKNEQDIRKLSSLMKQTPLTYIGLMTASLTMTGIPFLTAYFS KDLMMESAYKNSMLYWLAMMTATLTALYSTRLMYFSFLKNPQYYMTHSMSKNSETNHL MNSMMIMLTLMSMSTGYMTYTTLMQEQHPMMPQKLMLLPLFCSMLGAMIMMMIYPKNS TFKNFKKNMSLWKNFTSNAWNFNTIYNNIMTEKILNLIYKQSYKNMDKGLIEEIINNT TIKNMMKNSQNISKFQTTLLHTHLLTLILFYTSFSMYTQVSETQYSVMFWKNSSCSLK SCLGQTM*" CDS 210 . . 1558 /gene="cox1" /codon_star t =1 /trans l _ table=5 /product= "Cytochrome C Oxidase subunit 1" /translation="GFGNWFLPLLMGAPDMAFPRLNNMSFWLLPPSLFLLLSSSFVEN

YRMPLFIWAIFFTAFLLVLALPVLAGGMTMLLTDRNFNTTFFDPAGGGDPMLFQHLFW FFGHPEVYMLVMPAFGMMSHMIPYLTGKNQMFGYMGMVYAMASMGFLGFMVWAHHMFT MGMDVDTRAYFSAATMMMAIPTGMKMFSWIATMAAGTTRLETPMLWIMGFLFLFTMGG LTGIMCASASMDLMMHDTYYMVAHFHYVLSMGAVFGMFAATYFWYGKMTGLSLNELYS KIHFWSTFMGVNLTFFPQHFLGLAGFPRRYADFHDSFMTWNTLSSIGSMMSTTSAMFF LYMIMNSIYKSKSFKGWEDNYLATPSLEWMQENPPENHTFMELPHTSKFTQLLSNLK*

CDS 8785 .. 10063 /gen e ="nad2" /codon_start=1 / transl_table=5 /product="NADH dehydrogenase subunit 2 " /translation="TEMFLSLMLINLLMQSRWSTMKMISIMSTLSMMAYMYNNTLTLY SNTWTTFMQMWMFMGGLFLLKMINSHNTTIMTMSSSLMMASILLTTLKNWMMLYMTME MITITTLLLMSFNNKNAQSKEAS I KYL ILSAMSSTMLITGLLLASYSQNSYVVLLKTL LSSNNNLMLTMMLFKMGSAPFHIWMTDMYEGTKTKNLPMIILMPKMAMMSTILMFETN NNILLMCGMLSTAMGAMGAMNQKKMKRLLAYSSINNTGIMMMGLHMYTLPSIQASIAH IMMYTTTLTMILMTLQNTQNKKSLISEMTHNDNWYSKNKMILSTLLLSLSGLPPFPGF LSKWLIMSSTMKQQLLLTSTWMLLTNIPSTAYYFYTIIFSYFKKMYSKSYTMKEKTMK QYYMMASLTYPTLSILFHPQLILIPSWIASTTML" CDS 14508 . . 15704 /gene= "nad4" / codon_ star t=1 /transl_t a ble=5 /produc t = "NADH d e hydroge nase s ubunit 4 " /translation= "MNLMQTI MSPIMTLCILMLIPNYKKMTMETMSKLSTIFILFQTM YLHMETTENMKQMMLLFTSNSTINMTTLKIHHLCMDTLSTALMLLTTLLILTSILMSK KTIYHTHKSLYICLFMTMMMLFLTFSTSSLMMFYMMFESSIIPLIIMMALWGSRKEKM 48

RATYYFLLYTIMGSLPLFMTILSMHHTLYTFNTTTLYNTHMNTSTQMKLFMGLFLAFA MKTPLMPWHSWLPLAHVEAPAMGSVLLAGMLLKLGTYGFIRFTIPTLTQISKYFSPMI MTMSMLSMWLASMNSLRQNDMKRIMAYSSMAHMGMITAACFSLNPMSKTGAMMLMLSH GLTSSALFALMSYLYERHKSRLMKNFQGLSQTTPMLSTLIMITALAHMSTPGSINFMG EYLCLVG" CDS 13315 .. 14495 /gene="cob" /codon start=1 /trans1_table=5 /product="Cytochrome b" /translation="MMYNNHWPSERKSHFMFKAMNKILMDLPMPSNINYYWNLGSLLS FCLMIQLMTGMLLAMHYCPEIMHSFDSMAHMERNIYSGHMLRNLHANSASVFFLCVYL HMSRNIYHITWSNKMTWSMGITIYLTMMMTAFVGYVLPWGQMSFWAATVMTNLFSAIP YMGNSVVLWIWGSFSVSNPTLNRFYSLHYLFPFMLTSLIMMHMLALQTENHTNPMGNQ SEMDQMPFHHYFTTKDSQTIMFISVMIFFLATCIPYAFSDSENYMKANPLVTPTHMQP EWYFLFAYSMLRSIPNKLGGMLALVGSIAMLYLMLMLKQNNMKTTNHRPNLKLTTWLL SMTFMMLTWVGSKPAEAPYTTLSLMMTTWYFTSFLMLMPLHSITEKRTIYTSTMPHMM NEKSQ*" CDS 12024 .. 12977 /gene="nad1" /codon_start=1 /transl_table=5 /product="NADH dehydrogenase subunit 1" /translation="MMMNMMQATITFLPMLLTVAFLTLMERKILGSAQMRKGPNMVGF FGLLQPMADAVKLIMKENINPNKTNSLMFKLSPMMAMSMALTTWSLMPLHNNSPQSDT SMGIIMMLALSSLSVYTMLLTGWASNSKYSLLGSMRATAQMMSYEMSMGLMILSTVYI SSSLNLSIMTESQKYCWFMMPLFPAFTMLLISSLAETNRTPFDLTESESELVSGFNVE YSATLFTLLFLAEYTNILLMSTLMSMMFLGSTTMGTINNPLMLSMKIMLITYMFMWTR ATYPRMRYDQLMYLMWKSFLPMSLTLTTLMPSIIMMMNCLP*" CDS 7889 .. 8671 /gene="cox3" /codon_start=1 /transl table=5 /product="Cytochrome C Oxidase subunit 3" /translation="MKKYHPYHLVEASPWPIMGGCGALFLTCGSTLYFHYSHNTIMIA GFMIISTIMMTWWRDVMRESSFQGLHTMKVQLGLKLGMMLFITSEVLFFFSFFWAFFH SSLTPTMEMGANWPPEGMEALDPTAMPLLNTLTLLSSGMTMTWTHHSLMTNNKDNATK GLTWTILLGIFFTYLQMLEYYNSSFTISDSMYGSTFFVATGFHGAHVIMGTTFLTICR LRMNYNHFTTQHHVGFETSAWYWHFVDVVWLFLYTCMYWWGC*" CDS 5118 .. 5846 /gene="cox2" /codon_start=1 /transl_table=4 /product="Cytochrome C Oxidase subunit 2" /translation="MTTKLLLTSYFKDLPEKGQLNFQDPSSPMMEQIIMLHDYTMFIL MTVLMFMLWMLIKTTTHIMYWRNMSENTKLEIMWTTLPAIMLTIMAYPSLKLLYATDE PIEPELTMKSMGNQWYWSYEYSDYENKQMEFTSYMMPTDELTKGNNRVLEVDNRLIMP INTNMRMLMTAADVLHSFTIPSLGMKADAMPGRLNQVNFLASRPGLFYGQCSELCGAD HSFMPMVMESTNLKNYSYFMHTQT*" CDS 7002 .. 7729 /gene="atp6" /codon_start=1 /transl_table=5 /product="ATP synthase FO subunit 6" /translation="MNASYFNQFHLKKILYMKTSETIMSMSNLTLTLTMMLLLMITLN KSTKMLPNRNSMITHMLFTMTNNLTHEHMKEKRLSYNPFMLSMFTMFTAINLMGLLPY VFTTTSHMMITFSLSLTIIIKTTLSALMRHKSKFFSMLTPQSAPLLLAPFLVLMETTS YMTRAMSLGVRLAANMSAGHLLLAILSKFALDAMLTNHYILSLMPMTALFLMSMLEMM VALMQAYVFTLLTTIYLSDTMKLH*" CDS 6178 .. 6537 /gene="nad3" /codon start=1 /transl table=5 /product="NADH dehydrogenase subunit 3" /translation="MKQEEFIPMILMRIMSMSFSMILLTMSMMTSNNIPEAEKMSIYE CGFDPLKTSRLPFSMKFFLMGVLFLMFDLEISYIFPWSATMKETKLMSFMTMMLFLIM LTLGFMYEWLKKGLDWE*" CDS 6691 .. 6993 /gene="nad4L" /codon_start=1 49

/transl_table=5 /product="NADH dehydrogenase subunit 4L" /translation="MTHELISTTSMLMMLMSMYSMIMNFRNLIIVLMTMEMMLLALCL ILSTYTESLLLTFSTIMILQILTIAAAETAMGLSMLMTYYRMRGTITLKSLSLLRG*" CDS 1904 .. 2150 /gene="atp9" /codon_start=1 /transl_table=5 /product="ATP synthase FO subunit 9" /translation="MIYNMNNQNLMFCAKLMGAGAATMGVAGSGAGMGTVFGNLAIAY ARNPKLKQQLFTYAILGFAMSEAMGLFCLMMAFLILYGI* " CDS 1.. 208 /gene="cox1" /codon_start=2 /transl_table=5 / product="Cytochrome C Oxidase subunit 1" /translation="MFYQPQSYSHTMPNIRYICSLYSNFTKIINTTSIISNSNFISKR SFMQCHSNCPRFNNDIFLCHASLNW" gene 10064 .. 11959 /gene="nad5" gene 3298 .. >4935 /gene= "rnl" gene <1 .. 1558 /gene="cox1" gene 8785 . . 10063 /gene="nad2" gene 14508 .. >15704 /gene="nad4" gene 13315 .. 14495 /gene= "cob" gene 12024 . . 1297 7 /gene="nad1" gene 2396 .. 3296 /gene="rns" gene 7889 .. 8671 /gene="cox3" gene 5118 .. 5846 /gene="cox2" gene 7002 .. 7729 /gene= "atp6" gene 6178 .. 6537 /gene="nad3" gene 6691.. 6993 /gene="nad4L" gene 1904 .. 2150 /gene= "atp9" tRNA 5034 .. 5106 /ge ne= "trnS (ucu)" /product="tRNA- Ser" /anticodon tRNA 2325 .. 2395 /gene= "trnQ(uug)" / product="tRNA-Gln" /anticodon tRNA 11880 .. 11950 /gene="trnF (gaa)" /product="tRNA- Phe" /anticodon tRNA 1751. .1819 /gene="trnD(guc)" /product="tRNA-Asp" /anticodon tRNA 2259 .. 2327 /gene= "trnM(ca u)" /product= "tRNA-Met" /anticodon tRNA 65 52 . . 6620 /ge n e = " t r nW(uca) " / product="tRNA-Trp" /anticodon 50

tRNA 13114 .. 13182 /gene="trnN(guu)" /product="tRNA-Asn" /anticodon tRNA 2163 .. 2230 /gene="trnV(uac)" /product="tRNA-Val" /anticodon tRNA 6623 .. 6690 /gene="trnL(uag)" /product="tRNA-Leu" /anticodon tRNA 13244 .. 13311 /gene="trnY(gua)" /product="tRNA-Tyr" /anticodon tRNA 1540 .. 1606 /gene="trnT(ugu)" /product="tRNA-Thr" /anticodon tRNA 6109 .. 6175 /gene="trnK(uuu)" /product="tRNA-Lys" /anticodon tRNA 11951. .12017 /gene="trnC(gca)" /product="tRNA-Cys" /anticodon tRNA 13046 .. 13112 /gene="trni(gau)" /product="tRNA-Ile" /anticodon tRNA 12980 .. 13045 /gene="trnL(uaa)" /product="tRNA-Leu" /anticodon gene 1540 .. 1606 /gene="trnT(ugu)" gene 1751..1819 /gene="trnD(guc)" gene 2163 .. 2230 /gene="trnV(uac)" gene 2259 .. 2327 /gene="trnM(cau)" gene 2325 .. 2395 /gene="trnQ(uug)" gene 5034 .. 5106 /gene="trnS(ucu)" gene 6109 .. 6175 /gene="trnK(uuu)" gene 6552 .. 6620 /gene="trnW(uca)" gene 6623 .. 6690 /gene="trnL(uag)" gene 11880 .. 11950 /gene="trnF(gaa)" gene 11951. .12017 /gene="trnC(gca)" gene 12980 .. 13045 /gene="trnL(uaa)" gene 13046 .. 13112 /gene="trni(gau)" gene 13114 .. 13182 /gene="trnN(guu)" gene 13244 .. 13311 /gene="trnY(gua)" ORIGIN 1 tatattctac caaccacaaa gatataggca cactatacct aatattcggt atatttgcag 61 cctttatagg aacttcacta agattattaa tacgactaga attatctcaa acagaaactt 121 tattagaaaa cgatcattta tacaatgtca tagtaactgc ccacgcttta ataatgatat 51

181 ttttctttgt catgccagtc ttaattggcg gattcggaaa ctgattctta c ccctattaa 241 taggagctcc agatatggca tttccacgat taaataatat aagattctga c tattaccac 301 cttccctatt tctattacta tcatccagct tcgtagaaaa tggagtagga actggatgaa 361 ccttatatcc accactatcc aacatacaag cacattccgg aagaggcgtg gatctagtaa 421 ttttcagatt acatctcgcc ggatttcttc aattctaaga tcaattaatt ttctaacaac 481 cataataaat atgcgaacaa gagcaataac tgtatatcga atacctttat ttatttgagc 541 aatctttttt acagcatttc tactagtatt agcattacca gtactagcag gaggaataac 601 aatgctatta accgatcgaa actttaatac aaccttcttc gatcctgcag gaggaggaga 661 cccaatatta tttcaacatt tattttgatt ctttggacat ccagaagtat atatactagt 721 tataccagcc ttcggtataa tatcccatat aattccatat ttaacaggta aaaaccaaat 781 atttggatac ataggaatgg tttacgcaat ggcctctata ggattcctag gatttatagt 841 atgagcacac catatgttta caataggaat ggatgtagat actcgagcat atttctctgc 901 agccacaatg ataatagcaa ttcctacagg aataaaaata tttagctgaa tcgccacaat 961 agccgcagga acaactcgac tcgaaacccc catgttatga atcataggat ttttattctt 1021 attcaccata ggaggactta caggtattat atgtgccaga gcttctatag atttaataat 1081 acacgataca tactacatag tagcacattt tcactacgta ttatcaatgg gagccgtatt 1141 tggtatattc gcagccacat atttttgata tggaaaaata actggactaa gtttaaacga 1201 actttacaga aaaattcatt tctgaagtac attcatagga gtcaatttaa c attctttcc 1261 acaacatttt ctaggactag caggattccc tcgccgatac gccgattttc acgatagttt 1321 tataacatga aacacactaa gatcaatcgg atccatgata tcaaccacaa gtgctatatt 1381 ctttttatac ataatcataa attctatcta taaaagaaaa tcatttaaag gttgagaaga 1441 caactatctt gccacaccta gattagaatg aatgcaagaa aatcctccag aaaaccatac 1501 tttcatagaa ttaccacaca caagaaaatt cacacaactc ttatctaatt t aaaataaaa 1561 tacataactt gtaattatga catacaacac acactgtgat aagatatgcc acacctagaa 1621 acaacaacct acctacacag aatcacaaca ttttgactat tcctaattat attaactcta 1681 tgaagaacaa aaatacacta cacgacatat aaacaatata aaagaataaa c aaataggaa 1741 aaataaaata gaattataat ttaaaaaaaa gactaactgt cacttatgtc catagaagtt 1801 taactcttct taattcttac aaaaatacac tacacaacat aagaagaaaa tctaattaat 1861 ataatacaac aacacatcac ataactctac ataacaaata atttacaata tgaacaatca 1921 aaacttaata ttctgtgcta aacttatagg agcaggagct gctacaatag gagtagcagg 1981 aagaggagcg ggaataggaa cagtatttgg aaacttagca atcgcatacg ctcgaaatcc 2041 aaaactaaaa caacaactat tcacatacgc cattttagga ttcgctatat ctgaagcaat 2101 gggcttattc tgcctaatga tggctttctt aattttatac ggaatttaac attcacaaaa 2161 atcaaaaata atttaaaaaa aatcttgtct ttacaaaaca acaaataagg gccaatccct 2221 ttttttgaat ttaagaaaac acttaaaaga aaaaacgacc tttatagttt aaataaaaac 2281 ataagtctca tgaacttaaa ttgtggttaa ttacccacta aactagaaat tagtctaata 2341 gaaagactct gagccttgat ctctgcactg tagaatcata acctacattt ctaatcctat 2401 aaatttaaga ggaataagaa aataaaaatt atagacgtac atgctagcta aatagcaaac 2461 aagtgaaaaa taaaattact aatatataaa taataatttt ttaaagtaaa caatttattg 2521 cataaaacta tctaacagat cacatttaaa aaagcaacac ataacattgt gcaatatgta 2581 aaacatgaca cagcatcaat ccttataaca agaacaagca aagaatgtgc cagcagccgc 2641 ggccaaacat tctgtactaa ttttcaacac aacaacgtaa aaaagaaaag acaataatag 2701 aaaattgaac aataataaat agtaaaatat ataaataata attcaaccaa atctattttc 2761 taaaaaaaaa ttcttctaca ctaaagcata gatcaccaaa aggattagat accctcgtag 2821 cctatgccct aaataacaag aacaagtatg ctcgcaagac agaaacttaa taaatttgag 2881 agagtgagaa aactcaggga agcgtatact ttaattcgaa actacacgaa aatcttacct 2941 aaatttaaaa gtaagatatc tccgcatatt gtttttataa aaagaacaaa aatacactac 3001 acgacatatg ccggtcacca ccttcttcat atttagggcc agtaagcgcc acaaactatt 3061 atttttcact aatagttcct acaattgaaa aatataaagg agaacttgaa agtaattatc 3121 ccaaattaaa gtataatgaa acaaatataa aactgctaca aaccgcccgt caattcttaa 3181 gaaaaatata aaaaacaata ctaaaaaaaa agacaaataa attactacaa t ttacaaaca 3241 aaattaagaa taagtcgtaa caaggtagcc gtatctgaag atgcggctaa aaataaatga 3301 acttaatcat aaaaaaatcc tagatagact ctaaagtctc caacacaaca aataacaata 3361 aataactcac acaagaaaat aaaacattat cgacaataaa aatatttagc tagtaataga 3421 gaatgaaaac tacatacaag aataataaaa taattttaat acagagaaaa ctctaataaa 3481 cgaagagaca tttatataaa aagtactgaa aaggaaagaa taaacaaaaa ccccaatcta 3541 gaacatatta taaacaccca atattattcc ttttgcatca ggagcttatg agaaaaaatc 3601 ataaataaat tcaattaaat tcaaataaaa taaaaaccac taaatttatg aaacccgaaa 3661 ccttgtgatc taaccatgaa cagaaataaa aggaccaatt attaaatgtt gcaatatttt 3721 taaatgattc atgactagaa acaaaaagtt aatcgtacaa ggaaatacct ggttttcgaa 3781 gaaatatatt ttagtataac aaaaatcaga aagtataaaa aaataaaaat t ctgattcaa 3841 taagaaagaa agacaaacta accgtgaaaa gatagttagt ttaaatgaaa acagtcataa 3901 cctaaattaa aagtaaaaaa aatatttttt aaaatttaaa taacaaataa gtgggcttaa 3961 aaacagccac cttctaataa aaacgtaact gtttattaca aaaaagaaaa aaataaaaga 4021 tttataacaa aaacactata gattaggttt ttaaaaagat attcgaataa tactaataat 4081 ttaagcacta gtaaatacca cacacaccca aagtaaacta accggaatat aaaatttaaa 4141 gtaagaaaat ctaattaaat aaaacaaaaa ccgttcttta atccaactcc ggtgggatta 4201 agaaaattta ataattttaa ggaactcggc aaaacaggaa ttcgactgtt taccaaaaac 4261 atagccaata gaaaaaatta ttattggtga tgcctgctca atgattaaat aataaccact 4321 aaaaacaatt caatagctgc ggtaacacct gaccgtacaa aggtagcata ataaatcgcc 52

4381 tactaattat aggatagaat gaaggtaaaa cgaaaatcca actgtctcaa aattataaaa 4441 tttaaaatag aatgtgtgtg caaatccaac catgacggca gacgataaga ccctaggaac 4501 tttactaaaa acttaattaa acaaataata agtcgtaagt ttcgttgggg caacgatctt 4561 taaaaaagta actaagatta aataatcaca aaccacaatt tcattcaaat atacaagata 4621 agacccatta aataaaaaaa ttaatgataa cataataaaa gttccctagg gataacagcg 4681 ttatatcgtt tcaagagaac catccaaacg atgtttgcga cctcgatgtt gaattgctat 4741 atcctaaaat gtagccacat ttaaaggttg gactgtcctt ccattaaaat agcccatgat 4801 ttgagttcag accgaggcaa ctcaggtcag attctatcta ccgtcaaaac aaaaagacct 4861 gatataagta cgaaaggaaa atatctacta ccccatagca aaaccacgaa aaacaataaa 4921 gcttataaag cataaaactc tataacagaa aagatagcat aacgtatgca attgccttga 4981 aagctcttta taaggaataa caaccccttt ctcttcgaac tttaaaaaat aggaaaagtt 5041 agctaacaca gtaagcagac ggtattctaa accatcaaca ataagtgcaa c tcttttact 5101 tttcgtaatg gaaataaatg actacaaaac tactcct tac ctcctacttt aaagacttac 5161 cagaaaaagg acaattaaat tttcaagacc catcttctcc tataatggaa caaattatta 5221 tattacacga ctacaccatg tttattctaa taaccgtatt aatatttata ctatgaatac 5281 tcatcaaaac aaccacacac atcatatatt gacgaaacat gagagaaaac acaaaactag 5341 aaattatatg aaccacttta ccagcaatta tactaactat catagcatat ccttctttaa 5401 aactactata tgccaccgat gaaccaatcg aacccgaatt aaccataaaa agtataggaa 5461 atcaatgata ttgatcatac gaatattctg actatgaaaa taaacaaata gaatttactt 5521 cctacatgat acctaccgat gaattaacaa aaggtaataa ccgcgtactt gaagtagaca 5581 atcgactaat cataccaatt aataccaata tacgaatact aataaccgca gcagatgtcc 5641 tacactcatt tactattcct tctctaggaa taaaagccga cgcaatacca ggtcgactaa 5701 accaagtaaa tttcttagca tcacgaccag gattatttta cgggcaatgc tcagaattat 5761 gcggagcaga tcactctttc atgcctatag taatagaatc aacaaaccta aaaaactata 5821 gatactttat acacacacaa acttaaaagg aaaaaattaa gggattttag tttaaacaaa 5881 aacccttaat attgcttaat ttaaacatca ttaggaataa cacctataaa attcccaact 5941 actaatacta aaagatagat ttaaataaaa tcatttctcc tggggaagaa aaaaatacga 6001 gaaactcctc ggttctttta ataccggtag tttaaattaa aaaacactta aatttcgacc 6061 tttaaaaaaa taggagaaac cttttccgat acactaaatc caaaatataa agtataatta 6121 aataaataat ataagacttt tactctaaca tggtagatcc aactcctact ttaaaccatg 6181 aaacaagaag aatttatccc tataattctt a t acgtatca t aagaataag attttctata 6241 atcctactaa ccatatcaat aataactaga aataatattc ccgaagctga aaaaatatca 6301 atttatgaat gtggatttga tcctctaaaa acatcacgac taccattctc tataaaattc 6361 tttttaatag gcgtactatt cctcatattc gatttagaaa tctcctatat ttttccttga 6421 tccgctacta tgaaagaaac taaattaata agatttataa ccatgatatt atttttaatt 6481 atactaacac taggattcat atatgaatga ctaaaaaaag gattagattg agaataaaac 6541 atccttataa tgaaaaataa attaaattaa attataagac ttcaaatctt acaatactag 6601 taaaatctag ttttttcaga aatagatatg gtgaaataaa cacaattgac ttagaatcaa 6661 tctattgtag gccaacccta ctatctaaat atgacacatg aactaatctc aaccac taga 6721 atactaataa tattaataag aatatatagc ataatcataa attttcgaaa tttaattatc 6781 gtccttataa caatagaaat aatactacta gctttatgct taattcttag aacttacaca 6841 gaatcactat tactaacttt cagaacaatt ataattctcc aaattctaac tatcgctgca 6901 gcagaaacag caataggact cagaatacta ataacatatt atcgaatacg aggaacaatt 6961 acactaaaat ctctaagact cctacgtggc taaaaaataa atgaacgctt catacttcaa 7021 ccaattccat ttaaaaaaaa tcctctatat aaaaacaaga gaaacaatta taagaataag 7081 aaatctaaca ctaacattaa ctataatact tcttctaata attacattaa acaaaagaac 7141 aaaaatac ta cctaatcgaa attctataat cacacacatg ttatttacta taacaaacaa 7201 tttaacacat gaacacataa aagaaaaacg attaagttac aatcctttta tactaagtat 7261 atttacaatg ttcacagcaa ttaacttaat gggtttatta ccatatgtat t t accactac 7321 ctcacat ata ataatcacat tcaggctctc tctaactatt atcattaaaa caaccttaag 7381 agcccttata cgacataaaa gaaaattctt tagaatatta actccccaaa gcgcccccct 7441 attattagca ccattcttag tattaataga aacaaccaga tatataacac gagccatatc 7501 tctaggggtt cgactcgcag ctaatatatc agcaggcca t ttattattag caattttatc 7561 aaaatttgca ctagatgcaa tactcacaaa ccattatatt ttaagactca taccgataac 7621 agccctattt cttataagaa tac tagaaat aatggtagcc ttaatacaag catatgtatt 7681 tacattacta actaccatct atctttcaga taccataaaa cttcactaga aaaaataaga 7741 ttatacttaa cttaacacaa cttaaaaaac aatactatat aatagctaat taaacacaca 7801 cttaaaagga aaaattatta aatttttata catccactac tccaaataca ataaaaatta 7861 aaaacagaag t aaatatccc aaattaaatg aaaaaatatc accca tatca cctcgtagaa 7921 gcaagacctt gaccaattat aggaggctgc ggagcactat ttttaacatg tggaagcaca 7981 ctatactttc attattcaca taacacaatt ataattgccg gattcataat tatcagaact 8041 attatgataa cttgatgacg agacgttata cgagagtcct cctttcaagg attacacacc 8101 ataaaagtac aattaggatt aaaattagga atgatacttt ttatcacatc agaagtactt 8161 ttcttctttt ccttcttttg agcctttttt cacagaagat taaccccaac aatagaaata 8221 ggagcaaatt gaccacccga aggaatagaa gctctagatc caaccgctat acctttatta 8281 aacacactta cattactcag atcgggaata acaataacat gaacacatca ctctctaata 8341 accaataata aagataacgc cacaaaaggt ttaacatgaa caatcttatt aggaattttt 8401 ttc acatatt tacaaatatt agaatacta t a actcttcat ttaca atctc agattctata 8461 tatggtt caa ctttctttgt tgccacagga tttcacggag cccatgttat tataggaact 8521 acctttctaa ctatctgtcg attacgaata aattacaatc attttaccac ccaacatcac 53

8581 gtaggattcg aaacaagagc ctgatactga cacttcgttg acgtagtatg attattccta 8641 tacacctgca tatattgatg aggctgctaa cacacactat ataatataga aaatatataa 8701 caccacacac aaaaataatt taaaattcaa atccataata atatccaaca taacacttaa 8761 aagaaaaaat aataaatgag ctgaactgaa atattcctat cactaatact aattaatctt 8821 ttaatgcaat cgcgatgaag aacaataaaa ataattagta tcataagaac tctatcaata 8881 atagcctata tatataataa cactttaaca ctttacagca atacatgaac aacctttata 8941 caaatatgaa tatttatagg cggattattt ttactaaaaa taattaattc acataatact 9001 acaatcataa caataagaag aagtcttata atagcttcaa tcttattaac cacccttaaa 9061 aactgaataa tactatacat aacaatagaa ataattacaa tcacaacact actacttata 9121 agatttaaca ataaaaacgc acaaagaaaa gaagccagta ttaaatactt aattttaaga 9181 gcaatatctt caaccatatt aatcacaggt ttactattag caagatatag acaaaataga 9241 tatgttgtac tcttaaaaac tctacttaga agaaataata acctcatatt aacaataata 9301 ttatttaaaa taggaagagc accctttcat atctgaataa c tgacatata cgaaggaact 9361 aaaacaaaaa atttaccaat aattatcctc atacccaaaa tagcaataat aagaactatt 9421 ttaatatttg aaactaacaa taacatccta cttatatgcg gcatactttc taccgctata 9481 ggagccatag gtgctataaa tcaaaagaaa ataaaacgac tattagcata cagaagaatt 9541 aataataccg gtattataat aataggacta cacatataca cactacctag aattcaagca 9601 agaattgcac atattataat atacacaaca accctaacta taattttaat aactctacaa 9661 aatacacaaa ataaaaaaag attaattaga gaaataacac ataacgataa ctgatatagg 9721 aaaaataaaa taattttatc aacactacta ctatcacttt caggcctacc gccatttcca 9781 ggattcttaa gaaaatgatt aattatatct agaacaataa aacaacaact ccttttaact 9841 tcaacttgaa t actcttaac caatattccg agaacagcct attacttcta caccattatc 9901 tttagatatt ttaaaaaaat atattccaaa tcttacacta taaaagaaaa aacgataaaa 9961 caatactata taatagctag acttacatat cctacactaa gaattctatt ccacccccaa 10021 ttaatcttaa ttccaagatg aatcgccaga acaactatac taaatgtacc tattaataat 10081 aagaacaccc ctcataagct ttattttaac atttataaat caaaagcgaa tacgaccaaa 10141 taccattcaa acattagctt gcataatcat attattcgct tgaataaaca gcgtaaccat 10201 attctacgaa tgcataataa acgacacaca ctgtcaaaca aaaattatta attgaataga 10261 aatagtacat atgagaagaa aaataagatt acatatagac acattatcta gctccatgtt 10321 atttattatt acatctattt ctctatttgt tcacgtatat t c tcttcaat atatgaatga 10381 agatccacat aaacaccgat tcattagata tttatcatta ttcacatttt t tatgataat 10441 tctagtaaga agaaataact ttatacttct actaatagga tgagaaggag tcggaatttg 10501 ttcttattta ttaataaatt tctgatatac gcgaattcaa gcaaataaat caggaataaa 10561 agccataata attaatcgaa taggagacat agccttacta ataagaacca taatcattat 10621 aaaaaaattt ggaagaacaa aatttgaaaa cctcatccat atacaagaaa atatcaaaat 10681 acaatttaca ataaatagca t c tgtctact actacttata gctgctatag gaaaatcttc 10741 actgatagga ctacatattt gattaccaga tgcaatggaa ggacctacac caatttcagc 10801 cttaatacat gctgctacaa tggtaacagc aggaatcttc ctattaattc gatcttcttc 10861 tttattagaa gaaaggaaaa taagattaat aataacagct tgaatcggag caataacagc 10921 tttttttgct gctacaactg gattaagaca aaatgatata aaacgaatta t agcatattc 10981 aacatgtaga caactaggat atatggctct agctatcgga atctcaaaat acaatattgg 11041 actcctactc cttctcatcc tatattcttt aaaagattac tattctatag gagcaggaat 11101 cgccattcac accataaaaa acgagcaaga tattcgaaaa ttaagaagac ttataaaaca 11161 aactccactt acctatattg gtttaataac agcctcacta actataaccg gaattccatt 11221 t ctaacagcc tatttttcaa aagatttaat aatagaaaga gcctataaaa acagtatatt 11281 atactgacta gctataataa cagcaacact cacagcactc tattctacac gactcatata 11341 cttctctttt ctcaaaaatc ctcaatatta tataacacac tcaataagaa aaaataggga 11401 aacaaaccat ttaatgaata gaataataat tatactaaca ttaatgagaa taagaaccgg 11461 atacataacc tatacaactc taatacaaga acaacatcca ataataccac aaaaactcat 11521 actcttaccc c t attttgca gaatacttgg ggcaatgatt ataataataa tctaccctaa 11581 aaatagaact tttaaaaact ttaaaaaaaa tataagac ta tgaaaaaact tcactagaaa 11641 cgcttgaaat ttcaatacta tttacaataa catcataaca gaaaaaatct taaacttaat 11701 ctataaacaa tcttataaaa acatagataa aggattaatt gaagaaatta ttaacaatac 11761 caccatcaaa aatataataa aaaatagaca aaacattaga aaatttcaaa c aacattatt 11821 acacacacat cttttaacat taattctatt ctatacaaga ttttccatat atacacaagt 11881 ctcagaaact caatacagag taatattttg aaaaaataga agttgcaggt taaaatcctg 11941 tctaggacag acaatatagc ataataacaa tgcatcaatt tgcaaaattg aaaactgtaa 12001 acaattacta ttgtctaatt aaatgataat aaatataata caagcaacca tcacattttt 12061 accaatatta ctaacagtag cttttttaac tctaatagaa cgaaaaattt taggaagtgc 12121 acaaatacga aaaggaccaa atatagtagg atttttcgga ttactccaac caatagccga 12181 tgcagtaaaa ttaattataa aagaaaacat caaccctaat aaaacaaatt ctctaatatt 12241 caaactctcc cccataatag caataagaat agcactcacc acatgatctt taataccatt 12301 acataacaat tctccacaaa gagatacttc tataggaatt attataatat tagctttatc 12361 ttcattaaga gtttatacca t attacttac aggttgagcc agaaactcaa a gtacagtct 12421 cttaggttcc atacgagcaa ccgcacaaat gataagatat gaaatatcta taggattaat 12481 aattttatcc acagtttata tttcaagaag actaaattta agaattataa cagaaagaca 12541 aaaatattgt tgatttataa taccattatt tccagctttt acaatgctac ttatca gaag 12601 attagctgaa acaaaccgaa cgccattcga tttaactgaa agagaatctg aattagtctc 12661 cggatttaat gttgaatact ctgctaca tt atttacacta ttattcttag cagaatacac 12721 taacattcta ctaatgagaa ccttaataag aataatattt ttaggaagaa caactatagg 54

12781 aactattaac aatccactta tattaagcat aaaaattata ttaatcacat atatatttat 12841 atgaacacga gcaacttatc cacgaatacg atacgaccaa ttaatgtatt t aatatgaaa 12901 aagatttcta cctataagat taacactaac aacattaata ccaagaatta t tataataat 12961 aaattgttta ccataataat caatatagtg aaaacacaat aaatttaaaa cttatatatg 13021 tagacaacaa ctctactatt gataaagaaa atagtttaaa taaaaacttt atatcgataa 13081 tataacattg taaataaaat tacttttcta attctacagt agcttaaaca aagcatttaa 13141 ctgttaatta aaaaaatact tacaaaatca agtctgtaga gccaatagaa aaaataaata 13201 gacattttta aaagaaaaaa cgataataaa tctcactaat tcggccagat aatctaaata 13261 agatacttcg ctgtaaacga aataataaga aaataacctc tttctagcca accatgatat 13321 ataacaacca ttgaccatcc gaacgaaaaa gacattttat atttaaagcc ataaacaaaa 13381 ttctaataga cttacctata ccctcaaata ttaactatta ctgaaattta ggatcacttc 13441 taagattctg tcttataatt caattaataa ccggtatact attagctatg cattactgcc 13501 cagaaatcat acattctttt gacagaatag cccatataga acgaaatatt t attcgggac 13561 acatattacg aaatttacac gccaatagag cttctgtatt ctttttatgc gtatatctgc 13621 acataagacg aaatatttac cacattactt gaagtaacaa aataacatga tctataggta 13681 ttacaatcta cctcactatg ataataacag ctttcgtagg ctacgtatta ccgtgaggac 13741 aaatgtcatt ttgagccgcc actgtaataa caaatctatt ttcagccatt ccttacatag 13801 gaaattctgt agtactatga atctgaggaa gatttagtgt atctaatcca acacttaatc 13861 gattttacag attacactac ttatttccat ttatactaac cagactaatc ataatacaca 13921 tattagcctt acaaacagaa aatcatacca atccaatagg aaatcaaaga gaaatagatc 13981 aaataccatt tcatcattat ttcacaacta aagattcaca aacaattata t ttattagtg 14041 taataatttt ttttttagcc acatgcatcc catacgcctt tagagattct gaaaattaca 14101 taaaagcaaa tcctttagta acccctacac atatacaacc tgaatgatat t tcttatttg 14161 catattctat attacgatct attccaaata aattaggagg aatattagca ctagttggaa 14221 gaatcgctat actctactta atactcatat taaaacaaaa caatataaaa accaccaacc 14281 atcgaccaaa tttaaaatta acaacttgac tattatcaat aacatttata atattaacat 14341 gagttggtag aaaaccagct gaagctccat atactacatt aagacttata ataactacct 14401 gatattttac ctctttttta atactaatac cactacacag aatcacagaa aaacgaacaa 14461 tttacaccag tacaatacca cacataataa atgaaaaaag acaataaatg aacttaatac 14521 aaacaattat aagaccaatc ataacactat gtattttaat actaattcca aactacaaaa 14581 aaataacaat agaaactata agaaaattat ctaccatttt catcttattt caaacaatat 14641 atttacacat agaaacaaca gaaaacataa aacaaataat actattattt acaagaaact 14701 ccacaattaa tataacaaca cttaaaattc atcatttatg catagataca ctctctactg 14761 ccctaatgct attaaccact ttattaattt taacctccat tttaataaga aaaaaaacca 14821 tttatcacac acacaaaaga ctctacattt gcctatttat aaccatgata atactatttt 14881 taactttttc cacttcaaga ttaataatat tttacataat gtttgaaagc tcaattattc 14941 cattaatcat tataatagca ttatgaggct cacgaaagga aaaaatacga gcaacctatt 15001 attttctatt atacacaatt ataggatcct taccactttt tataactatt t taagaatac 15061 accacacatt atacacattc aacacaacaa cactctacaa cacacacata aatacatcaa 15121 cacaaataaa actatttata ggattatttt tagcattcgc tataaaaaca cctctaatac 15181 catgacacag atgattacca ctagcccatg tagaagcacc agctatagga tcagtactat 15241 tagcaggaat acttctaaaa cttggaactt acggattcat tcgattcact attccaacat 15301 taacacaaat ttctaagtac tttagaccaa taattataac aataagaata ttaagaatat 15361 ggctcgctag aataaacaga ctacgacaaa atgatataaa acgaattata gcatattcat 15421 ctatagccca catgggaatg attactgctg cctgcttttc cctaaaccct ataagaaaaa 15481 caggtgcaat aatattaatg ttaagccacg gactcacaag ctcagcatta ttcgcactaa 15541 tgtcctatct ttacgaacga cataaatctc gacttataaa aaactttcaa ggattaagac 15601 aaacaacacc tatattatcc acattaatta taatcacagc attagctcac ataagaaccc 15661 caggatcaat taatttcata ggagaatacc tctgcctagt agga II 55

LOCUS Docosaccus_macul 17143 bp DNA linear UNA 28-NOV-2010 DEFINITION Docosaccus maculatus mitochondrion, partial genome ACCESSION urn: local: . :1290238483812.44 VERSION KEYWORDS Docosaccus, Euplectell idae, mitochondrion, genome, mitochondrial SOURCE mitochondrion Docosaccus maculatus (spotted biscuit plate sponge) ORGANISM Docosaccus maculatus Eukaryota; Metazoa; Porifera; Hexactinellida; Hexasterophora; Hexactinosida; Euplectellidae; Euplectellinae; Docosaccus FEATURES Location/ Qualifiers rRNA 3607 .. 5204 /gene="rnl" /produc t ="large s ubunit r ibosomal RNA" rRNA 1985 .. 2880 /gene="rns" / product="small subunit ribosomal RNA" repeat_unit 13435 . . >13719 repeat_unit 12764 .. >12975 misc_feature 9657" 9658 /note="Genei ous type: Editing History Dele t i on" mis e f eatur e 9844" 9845 / note="Gen e i ous type: Editing History De l e tion" mise feature 9858" 9859 / note="Geneious type: Editing History Deletion" mise feature <13985 .. 14044 / note="Geneious type: repeat" mise- feature <12721 .. 12760 /note="Geneious type: r epeat" mise- f e ature <13359 . . 133 66 /note = "Ge n e i ou s type : repeat " tRNA 12478 .. 12545 / gene="trni(gau)" /product="tRNA-Ile" /anticodon tRNA 12405 .. 12471 /gene="trnL(uaa)" /product= "tRNA-Leu" / a nticod on tRNA 13367 . . 13431 /gene="trnY(gua)" /product= " tRNA-Tyr" /anticodon tRNA 12547 .. 12609 / gene="trnN(guu)" /product="tRNA-As n" / anticod on mise- f eature 9504 .. 1132 7 /note= "Gen e i ou s t ype : nad 5 " / gene= "na d5" / n o t e = "Gen e ious type : g e n e " gene 114 . . 1694 /gene="cox1" gene 8215 .. >9503 / gen e= "na d 2 " ge n e 14049 . . 1 5224 / gen e = "cob" g e n e 15949 .. >17073 / ge n e = "na d 4" g e n e 11453 . . 12457 / ge n e ="nad1" gen e 7423 . . 8205 /gen e= "c ox3 " gen e 2879 .. 3613 / gene= "cox2" gene 6697 .. 7422 /ge n e = "a t p6 " g e n e 15272 .. 15799 / ge n e = "na d6 " ge ne 5881 .. 623 1 /gen e= "na d 3 " 56 gene 6387 .. 6692 /gene="nad4L" gene 5283 .. 5504 /gene="atp9" tRNA 5513 .. 5588 /gene="trnV(cac)" /product="tRNA-Val" /anticodon tRNA 6316 .. 6386 /gene="trnL(uag)" /product="tRNA-Leu" /anticodon tRNA 1915 .. 1984 /gene="trnQ(uug)" /product="tRNA-Gln" /anticodon tRNA 1691 .. 1759 /gene="trnG(ucc)" /product="tRNA-G1y" /anticodon tRNA 11310 .. 11378 /gene= "trnF(gaa)" /product="tRNA- Phe" /anticodon tRNA 1780 . . 1847 /gene="trnD(guc)" /product="tRNA-Asp" /anticodon tRNA 1850 . . 1917 /ge n e= "trnM(cau)" /produc t="tRNA-Me t" /anticodon tRNA 5606 .. 5673 / gene="trnA(ugc)" / product="tRNA-A1a" /anticodon tRNA 15815 .. 15882 /ge ne="trnH(gug)" /pr oduct= "tRNA-His " / a nticodon tRNA 5677 .. 5743 / gene="trnP(ugg)" / product="tRNA-Pro" / anticodon tRNA 6249 .. 6315 /ge n e= "trnW(uga)" /produc t = "tRNA- Trp" / a n t i cod on tRNA 11380 .. 1144 5 /ge n e = " trnC (gca)" / product="tRNA-Cys" / anticodon tRNA 5814 . . 5877 / gene= "trnK(uuu)" / produc t = "tRNA-Lys " / a nticodon CDS 114 .. 1694 /gen e = "cox1" /codon_ start=1 / transl_tab1e=5 / produc t = "Cytochrome c Oxidase Subuni t 1" / tra n s l a tion= "MITRWLYSTNHKDMGTLYLIFGTFSAFMGTSLSMLMRLELSQTG TLLENDHTYNVMVTAHALMMMFFFVMPILMGGFGNWFLPLLTGAPDMAFPRLNNKSFW LLPPSLFLLLSSTFVENGVGTGWTLYPPLSNMQ SHSGGGVDLVMFSLHLAGLSSMLSS MNFLTTMLNMRAPGMTIYRTPLFMWAMLFTAFLLVLALPVLAGGMTMLLTDRNFNTTF FDPAGGGDPMLFQHLFWFFGHPEVYVLVMPAFGMISHMIPYLTGKKQMFGYMGMMYAM ASMGFLGFMVWAHHMFTVGMDMDTRAYFSAATMMMAMPTGMKMFSWMTTIAGGTVRLE TPILWVIGFLFLFTMGGLTGMMCASASMDLIMHDTYYMVAHFHYVLSMGAVFGMFAGF YFWYGKMTGLSLNELYSKIHFWTTFMGVNLTFFPQHFLGLAGFPRRYADFHDSFITWN TLSSVGSLMTTTSTLFFIMMMLDTMIKKKPFKGWEDNHLPTTSLEWVTENPPENHTFM 57

ELPHTSKFTQTKLMNNMT*" gene 1985 .. 2880 /gene="rns" gene 1691 . . 1759 /gene="trnG(ucc)" gene 1780 .. 1847 /gene="trnD(guc)" gene 1850 . . 1917 I gene=" trnM ( cau) " gene 1915 .. 1984 /gene="trnQ(uug)" CDS 2879 .. 3613 /gene="cox2" /codon_start=1 /transl_table=5 / product="Cytochrome c oxidase subunit 2" /translation="MNNTNNLWFFKDLPEKQQLNFQDPSSPMMEQMMMLHDYTMFILL TILMLMLWILIKITTTTLYWRDMNENTKLEIMWTTLPAMILTIIAYPSLKLLYATDES MEPELTMKSMGNQWYWSYEYSDYEQSKIEFTSYMLPTEELKTGDNRLLEVDNRLIMPI NTNMRMLMTAADVLHSFTMPSLGIKADAVPGRLNQVNFLSNRPGMFYGQCSELCGTNH SFMPMVMEATSLKNYSYFMHTETNKP*" gene 3607 .. 5204 /gene= "rnl" CDS 5283 .. 5504 /gene="atp9" /codon_start=1 /transl_table=5 /product="ATP synthase FO subunit 9" /translation="MFCAKLMGAGDATMGVAGSGAGMGTVFGNLMMGYARNPKLKQQL FTYAMLGFAMSEAMGLFCLMMAFLMLYGM*" gene 5513 .. 5588 /gene="trnV(cac)" gene 5606 .. 5673 / gene="trnA(ugc)" gene 5677 .. 5743 /gene="trnP(ugg)" gene 5814 .. 5877 /gene= "trnK(uuu)" CDS 5881 .. 6231 /gene="nad3 " /codon_ s t art=1 / transl_table=5 / product="NADH dehydrogenase subunit 3" /translation="MKQEEYTPMMMLLMSSMMFSTALMMTSTLTSKNTAEPEKLSMYE CGFDPLSTPRLPFSMKFFLMGMLFLMFDLEMSYMFPWCTTMKEMKWMSFMTMLLFLMM LTMGFIYEWLKKKD*" g e ne 6249 .. 6315 /ge n e="trnW(uga)" gene 6316 .. 6386 /ge n e = "trnL (uag)" CDS 6387 .. 6692 /gene="nad4L" / codon_start=1 /transl_table=5 /product="NADH d e hydrogenase subunit 4L" /translation= "MKQEMMVKTTSMMI LLMSMISIMMNFRNLIMFLITMEMMMLALC LMLSTHTEMTTYTMSTMMMLKMLTIAAAETAMALSMLTTYYRTRGTMSVKSLNLLRG*

CDS 6697 .. 7422 / gene="atp6" /codon_start=1 / transl_ table=5 /product= "ATP synthase FO subunit 6" /translation= "MNASYFDQFNMKKLLFMKSSEMMISMSNLTLMMSVIMMMLMTTT NNKMLPTRNSMMMQTLYTMSNNLTKEHTMGKHKTYMPMMFTMFMMFTTMNLTGLLPYV FTPTSHMMITF SL SLTMMMKTTMSAMITHK SKFFSMLTPNNAPL}~APFLVLMETTSY MTRAISLGVRLAANISAGHLLITMLSKFTLNIMVSSNILLSMMPMSALFLMSMLEMMV AMMQAYVFTLLTTMYLSDTMKLH* " CDS 7423 .. 8205 / g e ne= "cox3" 58

/codon_start=l /transl_table=5 /product="Cytochrome c oxidase subunit 3" /translation="MKKYHPYHLVEPSPWPMMGGCAILFLTSGSMLFFHYSHTTLLLM GTMMMMTMTMAWWRDVMREASFQGLHTHKVQMGLKQGMILFMASEVLFFFSFFWAFFH SSLVPTTEMGANWPPEGINALNPMAIPLLNTLTLLSSGMTITWTHHSMMSGSKKNSMT SLTWTMMLGMFFTSLQALEYYNSTFTMSDSIYGSTFFVATGFHGLHVLIGTTFLSVCW LRLKNNHFTKQHHMGFETSAWYWHFVDVVWLFLYTCMYWWGY*" CDS 8215 0 08400 /gene="nad2" /codon_start=1 /transl_table=5 /product="NADH dehydrogenase subunit 2" /translation="MIWTEMLMLTMSLKLMTQSKMKLMKTLNMMSIIMMMSYTYTHIN TLLKPTWTYFMQLWMMMS" CDS 8402oo9503 /gene="nad2" /codon_start=l /transl_table=5 /product="NADH dehydrogenase subunit 2" / trans l ation = "GMFLYKTAQTSNTINATMLGSMLITSMMLTTFKNWMMLYISMEL LTMMTLLMISMNSKNAQSKEASTKYLILSSLSSTMMITGMALTNYKMNSSSMMLKTLI MNNNLMISVLMFKLGSAPFHMWMTDMYEGTTTKNLPLMMLMPKIAMLSTLMTFETQHN MLLICGMLSTMMGAMGALNQKKMKRLLAYSSMNNMGMMLMGIHTYTLPSMQASITHMM MYTTSTSMMLMTLTHMYNNKQLMSETFQNDKMNKHQNMMISMLLLSLSGLPPFPGFLS KWLMMSSMMKQKFLMTSMWMLMTNMPATAYYLYTMMFSYFKTIKSNNNSMKTKNFKTN KYKMATLTYPTISMLMHPQTMLMPSWMTSTTMM" CDS 9504 0 011327 /gene="nad5" /codon_start =1 /transl_ table=5 / product="NADH dehydrogenase subunit 5" / translation="MYIMTMSAPLMSFMTAHTHTTKMKQKTMQNMTCLILLFSWINSM MMLYECMMNHTHCQTKLWNWMETEYFTCKMSLHTDMLSSSMLFTVTLISFFIHLYSTE YMKNDPHTLRFMSYLSLFTFFMLMLMSSNNYMFLLMGWEGVGMCSYLLMNFWYTRTLA NKAGAKAMMMNRMGDIALLMSTMMMLKKFGSTKFEVLTHTHESANMNSDYICLLLLLG AIGKSSLMGLHVWLPDAMEGPTPVSALMHAATMVTAGMFMLMRSSTMLEESKISLTIT AWMGVMTAFFASSMGMTQNDMKRMMAYSTCSQLGYMALAMGISKYSMSLLHLMNHAFF KSLLFLGAGMAMHTMSNEQDMRKLSNLMKYTPITYTSLLMGSLTMTGIPFLTAHFSKE QMMENTTTHSMLCTLAMITAALTAIYSTRLLYFTFMKQPQYLLPNKHNSHDTLKLTNM TLTLLTMSSMMMGYMTFTMMTTHMQHPMIPQYTKKLPILCTMMSSMMLMLLYHTHNHS KNINMQILMKNYLSNAWNFNTLYNNMMSEKLMHIAHTQSYKNTDKGMMEMMINENMNK FLQSSSNKISNTQSSKISKHMTTLMMGYMTLTMWTTTLETQ*" gene 11310 0 011378 /gene="trnF(gaa}" gene 11380 0 011445 /gene= "trnC(gca}" CDS 11453 0 o12457 /gene= "nad1" /codon_ s tart=1 / transl_table=5 /product="NADH dehydrogenase subunit 1" /translation="MMTKIMSTLLTLLPMMLSVAFLTLMERKMLGSSQIRKGPNMVGP YGMLQPMADAIKLIMKENNKPNKINLILFTLSPMMAISMALTTWSMIPMKNNSPQSDT KMGMMMMLALSSLSMYTILLTGWSSNSKYSLLGSMRATAQMMSYEMAMGLMMLTTMYL SSSLNLSIMTEAQQYTWYFLPLLPAFMMLMI SSLAETNRTPFDLTESESELVSGFNVE YSAMLFTLLFLAEYMNILLMSTMISIIFLGSSMMMNMNNPIMLASKIMLMTYLFMLMR ATYPRTRYNQLMDMMWKSFLPLSLSLTTLIPSMMLMMNSLSNQYSEKHNKLKIYKRSQ * " gene 124050 o12471 /gene="trnL(uaa} " gene 124780 01 2545 /gene= " trni (gau}" gene 125470 012609 /gene="trnN(guu}" gene 13367 0 013431 /gene= " trnY (gua} " CDS 140490 o15224 /gene="cob" /codon_start=1 59

/transl table=5 /product="Cytochrorne oxidase b" /translation="MLYNTKWEPKRKEIFMLKPLTKMFMDLPVPSNMNYYWNLGSLLS FCLMMQLMTGMLLAMHYCPETMYSFDKMTHMTRNMSSGYMLRNTHANSASVFSCVCTY TLVEMYTTMHEKTMQHEEMGMTMYLMMMMTAFTGYVLPWGQMSFWAATVITNLFSAMP YMGNTMVLWMWGSFSMSNPTLDRFYSLHYRLPFMLTTSMMMHMMGLHYENHSNPTGAQ SEMDIMPFHQYFLTKDLQTVMMLMMLMMLMTILMPYALTDPENFMKANPLVTPTHMRP EWYFLFAYSMLRSMPNKLGGMLALMSSMMMLYLILFLKQKYMTTGNHRPNIKLTAWLL SMTFLLLTWMGSAPAETPYTNLSLMITAWYFISFTMLLPTHNYKEKIMMHNIYPQNN*

CDS 15272 .. 15799 /gene="nad6" /codon start=1 /transl table=5 /product="NADH dehydrogenase subunit 6" /translation="MMFLLCMNMTMTSLMMTTTTSSMFSAMWLMMTFMNAALMLMMLE MEFMAILMIMVYMGAMAMLFLFTMMMLNLNTKIKKEEETHLIPLSMMIIILIMKSLES SNNKSMMEMTNNNNNNMETMSKLLYSEYSPWFLMSSMMLLISMMATITMMQKEEYTSM KQQLFTQLQRKMHNK*" gene 15815 .. 15882 /gene="trnH(gug)" CDS 15949 .. 17073 /gene="nad4" /codon_start=1 /transl table=5 /product="NADH dehydrogenase subunit 4" /translation="MNLMQTMMSSMMTLLTLIMLPSTHTNTLKNMSKMSILFMLTQTM YLHVKSMQINKLMMMMLTNNTQTNNTLMNMKGLCMDNTSTPLLMLTTMLMLTCMLMSE KNMYHTHKTLLMCLFSTMLTLFLTFTTSNMMFFYMMFESSMIPLMMMMGLWGSRKEKM RATYYFLLYTMTGSIPLFLSMLTLYTHTGTFNNITLTNTHTPLHMQLQLFMGLFLAFA MKTPLIPWHSWLPLAHVEAPGMGSVLLAGMLLKLGTYGFMRFTMPMLPDTSKFMSPLM MTISMMSMWLASINSLRQNDMKRMMAYSSMAHMGMITAACFSTNPMSTKGAIMLMISH GLTSAALFALVSFLYERHKSRLIKNFQGSSFTHQFYQDYY*" ORIGIN 1 cacacactgt ccagttcaaa gataatttta aaatatgcaa caccattcat aataaaagaa 61 atagtaatag aattcattta caacaacaca gaaaaaaaaa acactaaaca taaatgatta 121 cacgctgatt atattcaaca aaccataaag acataggtac tttataccta atctttggca 181 cattctcagc atttatagga acttctttaa gaatactaat acgattagaa ctatctcaaa 241 caggaacact actagaaaat gaccacacat acaacgtaat agtaacagcc catgctttaa 301 taatgatatt tttctttgta atgccaattc ttataggagg atttggaaac tgatttctac 361 ccttacttac aggagcccct gacatggctt ttccacgatt aaataacaaa agattctgac 421 tacttccacc atctctattt ctactacttt catctacctt cgtagaaaac ggagtaggaa 481 caggatgaac cctataccca ccattatcaa acatacaatc acattcagga ggaggggtag 541 acctagtaat attcagacta catttagcag gattatcctc tatactaaga tccataaact 601 tcctaacaac aatattaaac atgcgggccc cgggaataac aatttatcga acaccactat 661 tcatatgagc aatactattt acagcatttt tattagtatt agcccttccc gtactagcag 721 gcggaataac aatgctacta acagaccgaa actttaacac tactttcttt gatcctgccg 781 gaggaggaga tcctatacta ttccaacacc tattttgatt cttcggccac ccagaagtat 841 atgtactagt tatacctgcc ttcggtataa tttcccatat aattccttac ctaacaggta 901 aaaaacaaat attcggttac ataggaatga tatatgctat ggcatcaata ggattcctag 961 gattcatagt ttgagcccac catatgttta cagtaggtat ggacatagac actcgagcat 1021 atttttctgc agccacaatg ataatagcaa tacccacagg aataaaaata tttagatgaa 1081 taaccacaat cgcaggagga accgtacgat tagaaacacc tattctatga gttattggat 1141 ttctatttct attcacaata ggaggtctaa caggaataat atgtgctaga gcatctatag 1201 atttaattat acacgacaca tactatatag tagcacactt ccactacgtt ctttccatgg 1261 gagcagtatt cggaatattc gcgggatttt acttctgata tggtaaaata acaggattat 1321 ctctaaacga actatacaga aaaattcatt tctgaactac tttcatagga gtaaacctga 1381 ccttctttcc acaacacttt ctaggactag cgggattccc tcgacgatat gccgacttcc 1441 atgacagatt tatcacatga aacacactaa gatccgtagg atccttaata acaaccacaa 1501 gaacattatt tttcattata ataatactag acaccataat taaaaaaaaa ccattcaaag 1561 gatgagaaga caaccaccta ccaactacca ggctagaatg agtaacagaa aatcctccag 1621 aaaaccacac attcatagaa ctaccacaca caagaaaatt cactcaaacc aaactaataa 1681 ataacataac ataattataa tttaataaaa atatttgact tccaatcaat aaatgtaaac 1741 aacaacctta ctaattatat aatcaccaca cacaaaccta gaattataat ttaataaaaa 1801 gacaaactgt cacttatgtc cacagaagca caaccttctt aattctaccc attttatagt 1861 ttaaacgaaa acacaagtct catgaacttg aattgtgaca aatactcact aaactagaaa 1921 ttagtctaaa gaaagaccct gagctttgat ctccacattg tagactcgac ccctacattt 1981 ctaatcctat gattttaaga ggaaaaagaa aaaataatta tagatgtacg tgcaagctat 2041 aaagcaaata aataaattcc caacattact actataaatt aaaaataata atattattaa 2101 aacaaacatc ttacaagaat attaacaaca cataacattg tgcaatatgc aaacacatga 60

2161 cacagcataa attaaaaaca agaacaggcc aaaaatgtgc cagcagccgc ggctaaacat 2221 tttgtactaa ttttccaaaa tgtttataat aaaggaatcc ataaaaaaag aaatatgaac 2281 aaaaaaataa tagtaaaata cccaaaatac aacacaaccc caacaacaca atttctaaaa 2341 a a a agaccta ctaca ttaaa gcatagaaaa caaaaaggat tagataccct tgtagcctac 2401 gccataaaaa caacaaaaaa agt atgctcg caagacaga a acttaataaa tttgagagag 2461 ctaaaaaaat cagagaggcg tatgttttaa ttcgaaaata cacgaaaatc ttaccttcac 2521 cccacaagta aaatatcccc gtatattgct ttta taataa aaaaaaacaa attcccctac 2581 ataataaata ggaaatatac c ggtcaccac actctt tatg tgaagggcta acaagcgcca 2641 caaactatta atcaaaccta acagttctaa taccat gaaa aaacaaggag aatttgaaag 2701 taattattca taataaaaac ataatgaaac actaaaataa gctgttacaa accgcccgtc 2761 aattcttaaa aaacactaac aaccaaagaa agaaaaaccc ataataaata ataaaaacaa 2821 aaa aattaag aataagt c gt aacaaggtag c c gtaccc ga aggtgc ggct aaaca taaa t 2881 gaacaa c a c a aat a acctat gatttttcaa agatcttcca g a aaaaca a c aatt a aactt 2941 t c aagatcct t c ttcc c caa taatgga a c a aataat aata c t acatga tt a tacc atgtt 3001 cattttactc actatcctta tattaatact atgaattcta atcaaaatta c caccactac 3061 cttatattga cgagacatga acgaaaatac taaactagaa atcatatgaa c aacactccc 3121 tgcaataatc ttaacaatta ttgcatatcc ttctctaaaa ctactatatg c cacagacga 3181 aagaatagaa ccagaactaa caataaaaag aataggaaac caatgatatt g atcatatga 3241 atac t c t gat tacgaacaaa gaaaaattga attcacctc t tacatgttac c aactgaaga 3301 a c taaaaac a gga g a caatc g a ctcctag a agtagacaac cga ttaatc a t accaattaa 3361 c a cca acata cgaat attaa t aactgcagc agacg tacta cat t c a t t t a c aataccttc 3421 c ttaggaat c aaag cagatg c a g tacctgg tcgattaaac caagtaaact tcttaagaaa 3481 ccgacca g ga a t a ttttacg gaca atg ttc a g aac t atgc ggaacaa a cc actca tttat 3541 gccaatagta atagaagcaa caagcctaaa aaactataga tactttatac acacagaaac 3601 taataaacc t tagaaaaact aaaaagtctc aaaatacaaa aaataataac cactaaaaaa 3661 taaaacatta aacacacaat aaaaataaaa taatt agtaa tagagaatga acattaaaaa 3721 aaaacaataa gaacaattta aaaaagaaaa cctcataaaa agaaagactt t aataaaagt 3781 a c tgaa a agg aaaaacatat a c taatctaa cccacattat a a acactaaa t attattcc t 3 841 t ttgcatcag gatcttat g a gaaagaa tat a aataaatc t taaaatatgt aatacgaaca 3901 acaacaaa tt t atataac cc g a aaccttgt gatctaac ca tgaacagaaa t atagaact a 3961 a ttataaaat g ttgcaat a t t ttt aaacga ttt atggat a gaaacaaaaa g t taaacgaa 4021 caagg aaata actggtttt c gaagaaatat atttcagtat aaaaaatcag aaaat aataa 4081 aaataataat acaaactcta atacaaccac acacacaaac tagtcgtgaa aagatgatca 4141 gtttaaacga aaacagtcat aatctaaatt aaacaaataa aatataaaat acacacataa 4201 aaggtagact caaaaacagc caccctctaa caaaaacgta actgtttact acataacaac 4261 tttaaataaa aaatttataa caa acactaa ccaa gattag attaaaggta ttcgaataac 4321 cat aat aat t t aag cact ag taaat aacac a cataaccca a ggga a acaa accggtcttt 4381 aac caaact a atagaaaat a taaa taataa aaactataga c taaaaccgt c ccaaaatcc 4441 acct c t ggt a ggact aaaat aat aacaaaa tcttaaggaa c t c g gcaaaa tga ggact cg 4501 actgt t t acc aaaaacatag c t aat agaaa aagtta t tat t agtgatgcc tgct caatgg 4561 t tgaataata acaaataaca acgactaaat a gcc g cggta acacctgacc gtg caaagg t 4621 agcataataa accgcctact aattatagga tagaatgaag gtaaaacgaa gacccaactg 4681 tctcaagacc ctaaaattaa aaatagaatg tgtgtgcaaa tccacactaa aacggcaaga 4741 cgaccagacc ctaagaactt tacccaaaac ttcagccact taaaagttat aagtttcgtt 4801 g gggcaa cga t c tttaaaa c a gtaactaag a c t aat caat caca caacat a ttaa caaat 4 861 a a c ttcaaac aggtaaaacc cattaaggaa aaact taatg aaaaaaaat a aaagttcctt 4921 agggataaca gcgtcatatc gttctttaga aaatattcaa acgatgtttg cgacct cgat 4981 g t tgaattgc tatat cccaa aatgtaacca catttaaagg t tggactgt c c ttccatcaa 5041 aatagcccat gatttgagtt cagaccgagg caactcaggt cagattctat ctaccgttcc 5101 catcaaaatg aaattgatac aagtacgaaa ggaaaatatc taataccctc tagtaaagcc 5161 aagaaaaaca ttaaagttta taaaac ataa ctacaaccc a taaagaaaaa atactaaata 5221 aaaaacataa ccacac a c ac acatccacta acataaataa taaaaatatg aaca acaatt 5281 a t a tattttg t gccaaatta ataggagccg g a gatgct ac t a taggagt a gcagg c agag 5341 gagccggaat aggaactgta ttcggaaacc t aat aatagg atacgcacga aaccccaaat 5401 taaaacaaca a tta tttaca tacgccat ac t aggatttgc t atat cagaa gcaatgggac 5461 t a ttc t g t ct cat gatggca ttcctaatat tatacggaat ataaataat t aaccaaaaat 5521 aatttaaata aaatctt gtt t t cacaaaac aatacataag gataattcct t tttttgaaa 5581 ccacacacaa gtgcaaatat aaacagggat tttagtttaa tgaaaacact aacctt gcta 5641 attaaaaata tagactcctc cctataaatt ccataactaa aaatagatta aacaaat cat 5701 ttctcc t g gg aag a a aaaat acaaga aaat cttgttc ttt a a tatcagta g tttaataaa 5761 aaacaat aaa cct cgaact t aaaaat a t a t aaatcctct a t ctgataata aaat aaagca 5821 taattaaaaa ataatataag acttttaat c t aacattgt a gct ccctact gcttt aaaac 5881 atgaaacaag aagaatatac accaat aata atattattaa taa gaagaat aatat t t tct 5941 acagccctca t aataacatc aacactaacc agaaaaaata ctgctgaacc agaaaagtta 6001 agaatatac g aatgcggatt cgat ccatta agtacaccac gcctacct t t t t ccat aaaa 6061 ttttttttaa taggaatact attccttata t t tgatttag aaatatccta tatat t t ccg 6121 t gatgcacca ccataaaaga aataaaatga ataagattta taacaatgct attattccta 6181 ataatattaa caataggatt catttacgaa tgattaaaga agaaggacta gaatgagagt 6241 aaatattt tg aaaaataaat taaaaaaatt ataagacttc aaatc ttaac atactagcat 63 01 atctggtttt ttcagatag a tgtggt gaaa a t aaacacaa ttgacttag a a t caat a t cc 61

6361 tgtaggttat acccctacca tctaaaatga aacaagaaat aatagtaaaa acaactagaa 6421 taataatttt attaataaga ataattagaa ttataataaa cttccgaaac ttaattatat 6481 ttttaattac tatagaaata atgatactcg cattatgctt aatactaagc acacacacag 6541 aaataacaac atacacaata agaacaataa taatactgaa aatattaaca attgctgcag 6601 cagaaacagc aatagcctta agtatactaa caacttacta ccgaacacga ggcacaataa 6661 gagtcaaatc tttaaacctt ttacgaggat aaacaaatga atgcttctta ctttgatcaa 6721 ttcaacataa aaaaattatt atttataaaa tcaagagaaa taataattag aataagaaat 6781 ctaactttaa taataagagt tatcataata atactaataa caaccactaa caataaaata 6841 ctccctaccc gaaattctat aataatacaa actctataca caataagaaa caacctaaca 6901 aaagaacaca caataggaaa acacaagaca tacataccaa taatattcac aatatttata 6961 atatttacaa ctataaatct aacaggatta ctcccatacg tatttacacc aacatcacac 7021 ataataatca catttagact atccttaac t ataataataa aaactactat aagagctata 7081 attacacaca aaaggaaatt ttttagaata cttacaccaa ataacgcccc cttaatatta 7141 gccccattcc tagtattaat agaaacaaca agatatataa cccgagcaat ttctttaggt 7201 gttcgactag cagcaaacat ttctgcagga catctactta ttactatact atcaaaattc 7261 accttaaaca tcatagtaag cagaaatatt ttattaagaa taataccaat aagagcctta 7321 tttctcataa gaatactaga aataatggta gcaataatac aagcttatgt attcacactc 7381 ttaacaacaa tatacttatc tgacacaata aaactccatt aaatgaaaaa ataccaccct 7441 tatcatctag tagaaccaag accatgacct ataataggag gctgtgctat cttattttta 7501 acaagaggaa gaatactatt tttccactac tcacacacca cactcttatt aataggaaca 7561 ataatgataa taacaataac aatagcctga tgacgagatg taatacgaga agcctcattc 7621 caaggactac acacacataa agtgcaaata ggattaaaac aaggaatgat t ttatttata 7681 gcttctgaag tactattttt cttctctttt ttttgagctt ttttccacag aagattagtc 7741 ccaacaacag aaataggagc taactgacca ccagaaggaa ttaatgcttt aaatccaata 7801 gccattcctt tactaaacac actaacactt cttagatcag gaataactat cacatgaaca 7861 caccactcta taatatcagg aagaaaaaaa aactctataa caagactaac atgaacaata 7921 atactcggaa tattctttac atccctacaa gcattagaat actacaactc tacatttaca 7981 atatcagact caatttatgg ctcaaccttt tttgtagcaa ccggatttca tggattacat 8041 gtattaattg gaacaacctt tctttccgtc tgttgactac gattaaaaaa caatcacttc 8101 acaaaacaac accacatagg attcgaaaca agagcatgat attgacactt cgtagacgta 8161 gtctgattat ttctatacac ctgcatatac tgatgaggat actaaaacca ttaaatgatt 8221 tgaacagaaa tacttatact tacaataaga ctcaaactaa taacacaatc gaaaataaaa 8281 ctaataaaaa cattaaacat aataagaatt attataataa taagatatac ctacacacat 8341 attaacacac tactcaaacc cacatgaaca tatttcatac aactctgaat aataataaga 8401 tggaatattc ctatacaaaa cagcacaaac aagaaacact attaacgcaa caatattagg 8461 aagaatatta attacatcaa taatactcac cacatttaaa aactgaataa tactatatat 8521 ctcaatagaa ctactaacaa taataacact attaataatt agtataaata gtaaaaacgc 8581 tcaaagaaaa gaagctagaa caaaatattt aatcctaaga agcctatcat caactataat 8641 aattacaggc atagcactta ctaactataa aataaacaga agaagaataa tattaaaaac 8701 attaattata aataacaacc taataatcag agtattaatg ttcaaattag gaagagcccc 8761 tttccacata tgaataacag acatatacga aggtacaacc actaaaaacc tacctttaat 8821 aatactcata ccaaaaattg ctatattaag aactcttata acattcgaaa cacaacacaa 8881 catactatta atctgcggta tactttcaac aataataggg gctataggag ctttaaatca 8941 aaaaaaaata aaacgacttc ttgcctacag aagaataaat aacataggaa taatactaat 9001 aggaatccac acatacaccc taccaagaat acaagcctct attacacata tgataatata 9061 cactacaaga actagaataa tattaataac acttactcac atatataata acaaacaatt 9121 aataagagaa acatttcaaa acgacaaaat aaacaaacat caaaatataa taatttcaat 9181 actattactc tcattatcag gactccctcc attccccgga tttctaagaa aatgactaat 9241 aatatcaaga ataataaaac aaaaattttt aataacatct atatgaatat taataaccaa 9301 c a t acctgca acagcatatt acctatacac aataatattt aggtacttta aaactattaa 9361 gagaaataat aataggataa aaacgaaaaa ttttaaaaca aataaatata aaatagcaac 9421 actaacatac ccaactatta gaatattaat acacccacaa acaatattaa tgccaagttg 9481 aataaccaga acaacaataa taaatgtaca ttataacaat aagagccccc ctaataagat 9541 tcataacagc acacacacat acaacgaaaa taaaacaaaa aacaatacaa aacataac tt 9601 gtcttattct attattctct tgaatcaata gaataataat actatatgaa tgtataataa 9661 accacacaca ctgccaaacc aaattatgaa actgaataga aacagaatat ttcacctgta 9721 aaataagact acacacagac atactttcta gttctatgct atttacagta acactaatct 9781 cattctttat tcacctctac tcaacagaat acatgaaaaa cgacccacac acattacgat 9841 ttataagata tctatcctta ttcacattct tcatgctaat actaataagt agaaacaatt 9901 atatattttt actaatagga tgagaaggag taggaatatg ctcttatcta ttaataaatt 9961 tttgatacac ccgaacacta gctaacaaag ccggagcaaa agctataata ataaaccgaa 10021 taggagatat c gcactacta ataagaacca taataatatt aaaaaaattc ggcagaacaa 10081 aatttgaagt cttaacacac acacacgaaa gtgcaaatat aaacagagat t acatctgtc 10141 ttcttttact actaggagcc attggaaaat cttctctaat aggactacac gtatgattac 10201 ccgatgcaat ggaaggaccc actccagttt ccgctctaat acacgccgct accatggtaa 10261 cagcaggaat attcatactc a t acgatcat ccacaatact agaagaaaga aaaattagat 10321 taactatcac agcttgaata ggagttataa cagcattttt cgccagaaga ataggaataa 10381 cacaaaacga tataaaac ga ataatagcat actcaacatg tagacaatta gggtatatgg 10441 ctctagccat aggaatttca aaatactcaa taagactctt acatctaata aaccacgcat 10501 tcttcaaaag acttctcttc ttaggagccg gaatagcaat acacacaata agaaacgaac 62

10561 aagatatacg aaaactaagt aacctaataa aatac acccc aattacatac acaagtctac 10621 taataggttc attaaccata accggaattc cttttctaac agctcatttc tcaaaagaac 10681 aaataataga aaacacaacc acacacagaa tactctgtac attagc aata attacagcag 10741 c attaactgc cattta ttct acacgtc ttt tatatttcac tttc ataaaa caaccacaat 10801 atttattacc taataaacat aactcgcacg acacactcaa attaactaat ataaccctta 10861 ccttactaac aataagaaga ataataatag gatatataac attcaccata ataaccacac 10921 acatacaaca ccctataatt ccacaatata caaaaaaact acctattctt tgcacaataa 10981 taagaagaat aatactaata ctcttatacc acacacacaa ccacagaaaa aatattaata 11041 tacaaatttt aataaaaaac t acctaagaa atgc c t gaaa tttcaatacc ttatataaca 11101 acataataag agaaaaatta atgcatatcg cacacacaca atcctacaaa aatacagaca 11161 aaggaataat agaaataata atcaacgaaa atataaacaa attcttacaa agaagaagaa 11221 aca aaattag aaac acacaa tcatccaaaa tttc taaaca tataacaacc ctaataatag 11281 gatatataac acttac a a ta tga acaac a a ccctagaaac t caa tagagt g ttattttga 11341 aaaaataaaa gaaatagatt caaatcctat ctaaggtaga caataaagca taaaaaatgc 11401 atcaatttgc aaaattgaaa actgtgaaaa tactcactac tgtctaacac tcatgataa c 11461 aaaaattata agaactttac taacactctt accaataata ctaagagtag ccttcctaac 11521 attaatagaa cgaaaaatac ttggaagaag acaaatccga aaaggcccaa atatagtagg 11581 accctacgga atattacaac caatagc aga tgcaattaaa cttattataa aagaaaacaa 11641 taaacccaat aaaatcaatt taattctttt cac attatct cctataatag ccatcagaat 11701 agcacttaca aca tga tcta taatccc a at a aaaaa c a a c tccccacaaa gagatacaaa 11761 aat aggaatg a t aat aat a c tcgcc ttatc ttcattaag a a t a t a c acc a tctta ttaac 11821 aggttg a t ct agaaattcca aatatagact act a ggtt ca atacgagcaa ctgcacaaat 11881 gataagatac gaaatagcaa taggac taat aatatt aac a acaatatatt tatc aagaag 11941 attaaacctt agaattataa cagaagcaca acagtac aca tgatatttct tac ctttatt 12001 accagcattt ataatgctaa taatcagaag actagcagaa actaaccgaa c accttttga 12061 cctcacagaa agagaatcag aactagtatc tggtttcaac gtagaatact c agcaatatt 12121 atttacactt ctctttctag ccgaatacat aaatatt ctc ttaatgagaa c cataattag 12181 aatcatcttt ttag gaagaa g aatgataa t aaacataa a c aaccctatt a t a ttagcaag 12241 aaaaatta tg t t aat aacat atctattcat a ttaatac ga gcca c atatc cccg aacacg 1 2 301 atacaatcaa ttaatggata taat atgaaa aagatt cctt ccat taagac t a t catt aac 12 3 61 aaccctaatt ccaagaataa tact aataat aaact cacta tcaaatcaat atagtgaaaa 1242 1 acacaataaa c tta a aattt ataaacgta g a caataattc t acttttgat a t caaaa c ag 12481 aaaatagttt aacaaaacac catactgata atatgatact gtagataata attctacttt 12541 tctaactcta tagtagctta aattaaagca tttaac tgtt aattaaaaaa tactaaaaag 12601 tctacagaga taaaaacata atataataat tact tat tat caaattaaat tattattaag 12661 c ccacaacac ttacaatata aaacatatgc taaaat g c ta gataatctaa agaaagatat 1272 1 ttc g c tgta a acgaaaatat a aaacatacc tttccggcca a a aatgttac t taata ctaa 12781 acgagaacca aaacgaa aag aaattt t cat a ttaaaac c a ttaag aaaaa acta c accaa 1284 1 ctcct tac tg ccaaaaaaca t taaacacta ttactgaaat t ttag attt c tattaat act 1 2901 a t acct aaca atacat ttaa t aaccacaat act attaat a ataaattacc a c ccaaaaat 1 2961 aagacat acc tttaacgaaa t aactctttt t aaacaacaa ctat acagcc acaga a t a tc 1302 1 catttttttc tttcgtctat atttatattt taaacaaacc acacacaaca taacatgaaa 1 3 0 81 aac agaaata acaatatacc taataagaat atta ataac a tataaaatat attcattacc 1 3141 cttaaaaaaa a tatttggtt g aatta ttat taaac t cata acaactctaa c acgaaagga 1 3201 aactttta tt gcaaaa acag g a ga c a aata cctta t aata ctta a a a aga c a ccaa a cca 1 3261 agaa aaagag aaaaaacatt aaaaact a a a act atttcga a aatatagga ttatta aaca 13321 aataaaat ac aattatttaa aaacacaaca c acactaa ct ataaa aggct a gat aat cta 13 381 aagaaagat a tttcgctgta aacgaaaata taagacatac c t ttccagcc aaaaatgcta 13441 tataacatt a aacacacacg aaagg aaatt ttcactttaa aaccattaag aaaaaactac 1 3501 accaatcttc taccaccaac taataaaaat tactgaaact tcaaatatat attaatactc 13561 tatctaaaca ca c a c tta gt a a caataa ta ttactaataa taaattacta c ccagaaaga 13621 a a atattctt ttaacaa a at a ttaga tata a a a c a ag a ca tatcatgaga acttatacaa 13681 g a a tacatgt acgcac a t a g aatatcc a tt t t t tttcttc acttatgtat a c a actc a aa 13741 caaag cataa acaacaat ac agaaaacaac cat actccct t agaaacagc a ttaaat a t t 1 3801 tact taatat taat aacagc ttgcatatta tacttattac cctgaaaaaa tat a tttact 1 3861 tgaatcatta ttaaactcat aacaagacta acacaaaagg aaact tttat cgtaaaaaca 1 3921 aaatttcat a cccttatcat aaca agcgaa aaaatatta a accaat gaaa a a agaaat ag 13981 agctagat aa tctaaagaaa gatatttcat tacaaacgaa aatataagac atacctttcc 14041 ggccaaaaat gctatataat actaaatgag aaccaaaacg aaaagaaatt ttcatattaa 14101 aaccattaa c caag atattc atagactt a c cagta ccgtc caatataaac tacta ctgaa 141 61 a t c ttgggt c cctac t aaga ttct gtct aa t a atacaatt aat aaca gga a t actattag 14221 caatgcat ta ttgcccagaa a caat a t act cttttgacaa aataacacac a t aacacgaa 14281 acat a t catc aggatacatg cttcgaaaca cacacgccaa cagagcatcc gttttttcct 1 4 341 gtgtgtgtac ttacacatta gtcgaaatat ataccacaat acatgaaaaa accat acaac 1 4401 atgaagaaat aggaataaca atatacttaa taatgataat aacagctttc acaggatacg 1 4461 tactaccctg aggacaaatg tccttctgag ccgctactgt aattacaaat ctattctcag 14521 caatacctta cataggaaat actatagtcc tatgaatatg aggaagcttc agaatatcaa 14581 accccacact agatcgattt tatagcttac actaccgact tcctttcata ctaaccacaa 14641 g a a taataat acatataata ggtttacact a cgaaaacca ctcaaatccc acaggagca c 14701 aaagagaaat agacat cat a cctt t t cat c aat actt tct aacaaaagac c t acaaacag 63

14761 taataatatt aataatatta ataatattaa taacaatcct cataccttac gcactcaccg 14821 acccagaaaa tttcataaaa gcaaaccctt tagtaacacc cacacacata cgaccggaat 14881 gatatttctt atttgcatac tcaatattac ggtcaatacc aaacaaacta ggaggcatat 14941 tagcccttat aagaagtata ataatactat acctcattct atttctaaaa caaaaatata 15001 taactacagg aaaccatcgg ccaaacatta aacttacagc atgacttcta agaataacct 15061 tcttattact aacctgaata ggaagagccc ctgccgaaac accttatacc aaccttagac 15121 taataatcac agcttgatac ttcatttcat tcacaatact attacccaca cataactata 15181 aagagaaaat tataatacac aacatttatc ctcaaaataa ttagaaatag aaaatgaaca 15241 ttaaaaaccc cctgtactaa caacacctta aatgatattt ttactttgca taaacataac 15301 aataacaaga ctaataataa ccacaacaac aagaagaata ttcagagcca tatgactaat 15361 aataacattc ataaacgctg ctttaatatt aataatactc gaaatagaat tcatagcaat 15421 cctaataatc atagtatata taggcgccat agccatacta tttttattta caataatgat 15481 gctcaactta aacactaaaa ttaagaaaga agaagaaaca catctaattc cactaagaat 15541 aataattatt attctaatca taaaaagact agaaagaaga aataataaaa gaataataga 15601 aataacaaat aacaacaata ataatataga aacaataagt aaactactat attctgaata 15661 tagaccttga tttctaataa gaagaataat actattaatc agaatgatag caacaatcac 15721 tataatacaa aaagaagaat atacgagaat aaaacaacaa ttatttacac aacttcaacg 15781 aaaaatacac aataaataat aacaaatata tactataaat atagcttaac aaaagctaag 15841 acttgtggag tcttccatac aaaatcaaat ttgttattta taaccttatc ggtaatttaa 15901 taaaaataca taaaattaaa aataaaatcg aaatacagcc ccgataatat gaatctaata 15961 caaacaataa taagaagaat aataacacta ctaacactaa ttatattacc tagcacacac 16021 actaacacac taaaaaatat aagcaaaata tccattctat tcatattaac acaaacaata 16081 tacctacacg ttaaaagaat acaaatcaat aaactaataa taataatact cacaaacaat 16141 actcaaacta ataacacact aataaatata aaaggtcttt gtatagacaa cacatccaca 16201 cctttactaa tattaacaac aatactaata ttaacatgca tattaatgag agagaaaaac 16261 atataccaca cacataaaac attattaata tgtctattca gaacaatatt aacattattt 16321 ttaacattta ccacatccaa tataatattc ttttacataa tgtttgaaag atcaataatt 16381 ccactaataa taataatagg attatgagga tcccgaaaag aaaaaatacg agccacatat 16441 tattttctat tatacacaat aactggatct attcctttat ttttaagaat attaacctta 16501 tatacacaca caggaacttt caacaacatc acattaacta acacacacac gccattacac 16561 atacaattac aactatttat aggactattt ttagcctttg ccataaaaac acctttaatt 16621 ccatgacaca gatgacttcc tttagcacac gtagaagccc ccggaatagg atccgtacta 16681 ctagcaggaa tactactaaa actaggaaca tacggcttta tacgattcac cataccaata 16741 ctaccagaca ctagaaaatt catatcccct ttaataataa ccattagaat aataagaatg 16801 tgattagcaa gaattaacag attacgacaa aacgatataa aacgaataat agcatactct 16861 tctatagcac acatgggaat aatcacagca gcatgtttct caacaaaccc tataagaaca 16921 aaaggagcta ttatattaat gatcagccac ggattaacaa gagcagcatt attcgctctc 16981 gtttcatttc tatacgaacg acacaaatca cgactcatta aaaatttcca agggagaaga 17041 tttacacacc aattttatca agactactat taataacaat tctagcacac atgagaaccc 17101 caggatctat aaattttata ggagaatacc tctgcctagt agg II 64

APPENDIXB

GLOSSARY OF TERMS USED IN HEXACTINELLID TAXONOMY 65

Taxonomic descriptions require specific nomenclature that, while widely known within the taxonomic community, requires definitions for the newly-initiated reader. The definitions listed below are only those required for understanding the descriptions of Bathydorus laniger and Docosaccus maculatus, presented in this thesis. Complete dictionaries and thesauri are available from Tabachnick and Reiswig (Dictionary of Hexactinellida, in: Hooper, J. N. A. et al. (Ed.). Systema Porifera: a guide to the classification ofsponges. 2002, 1224-1230) and Boury-Esnault and Riitzler (Thesaurus of Sponge Morphology; Smithsonian Contributions to Zoology, 1997, 596, 64 pp) .

List of terms atrial surface - surface of the sponge from which exhalant water is released. atrialia - spicules lining the atrial surface. axial filament -proteinaceous filament in the center of all spicules. Hexactinellid spicules have triaxial filaments, with a ray of the filament lining each spicule ray. basalia - long prostal spicules that are directed downward, serving to anchor sponges into soft sediments. basiphytous- a method of attachment in which a hard, fused plate attaches a sponge to a hard substrate. choanosome - the region in a hexactinellid between the dermal and atrial surfaces, and within which choanomere chambers exist. dermal surface - surface of the sponge from which inhalant water is drawn. dermalia- spicules lining the dermal surface. diactin- megasclere spicule with two long rays, appears as a long rod. The other four rays may be nonexistent or vestiges may remain as tubercles in the center of the spicule. discohexasters- six-rayed microscleres with discoidal, or cupped, tips. distal ray - ray of a megasclere that is directed away from the choanosome. floricome- six-rayed microsclere with S-shaped, slightly spiny secondary rays. Each secondary ray with a recurved claw at the tip. 66

hemihexaster- six-rayed microsclere that is similar to a hexaster, but with at least one primary ray diverging into a single secondary ray. hexactin - megasclere with six rays of equal or unequal length. hexactine- adjective form ofhexactin. hexaster- six-rayed microsclere with 2-3 secondary rays branching from each primary ray. hypoatrial- the region just proximal to the atrial surface, often in reference to "the spicules that comprise the hypoatrial region." hypodermal - the region just proximal to the dermal surface, often in reference to "the spicules that comprise the hypodermal region." lophophytous -a method of attachment in which loose tufts of basal prostalia project into sediments, anchoring a sponge to a soft substrate. marginal prostalia - prostal spicules that protrude from the margin of a sponge, creating a fringe or ring around the margin of the sponge. megasclere- a large spicule of known structural importance to the architecture of a sponge skeleton. microsclere - a small spicule, usually 100 Jlm or less, with no known structural importance to the architecture of a sponge skeleton. osculum - a hole or pore on the atrial surface from which processed water is exhaled back into the environment. ostium -the smallest hole on the dermal surface, between 20 and 100 Jlm , into which water flows on its way to choanomere chambers. oxyoidal- relating to the tips of microscleres. Oxyoidal tips taper sharply to a point. oxyhemihexaster- a hemihexaster (six-rayed microsclere with at least one primary ray that diverges into a single secondary ray) with oxyoidal (pointed) tips. oxyhexactin - a hexactine microsclere with oxyoidal (pointed) tips. oxyhexaster- a hexaster with oxyoidal (pointed) tips. parietal oscula - holes that protrude through the entire body of a sponge. pappocome - a six-rayed microsclere with brush-like secondary rays. 67 pentactin - megasclere with five rays of equal or unequal length. pinole- a specialized hexactine megasclere, in which the proximal ray is much longer than other rays and contains many curved spines directed toward the tip. pleural prostalia- prostal spicules projecting from the dermal surface. In tubular and saccular sponges, pleural prostalia project from the dermal surface and do not anchor into the sediments. In plate-like sponges, pleural and basal prostalia are both directed toward the seafloor and are assumed to be synonymous. primary ray- in microscleres, the ray from the center of a spicule to the first divergence, when it branches into secondary rays. prostalia - long spicules that project from the dermal, basal, or marginal regions of a sponge, creating a pubescence or spicule "fuzz." In the basal region, prostalia are responsible for lophophytous attachment to soft sediments. proximal ray - ray of a megasclere that is directed toward the choanosome. sacciform - in gross morphology, sac-shaped. secondary ray- in microscleres, the ray that projects from a primary ray. spicule- the skeletal unit in hexactinellids. stauractin- four-rayed megasclere, either with all rays projecting in the same plane (forming a cross) or all directed slightly downward. tangential ray -ray of a megasclere that is parallel to the dermal or atrial surface. tubular- in gross morphology, tube-shaped. 68

APPENDIXC

MEASUREMENTS OF SPICULES FOUND IN BATHYDORUS LANIGER 69

Choanosomal Basal Gastral hexactin Hypodermal pentactin Dermal stauractin Oxyhemihexaster diactin diactin

Ray Tangential Tangential Proximal Proximal Ray Length Primary Primary Ray width Ray width Length (mm) Diameter length ray length ray width ray length ray width length (mm) ray length ray width 1 80.9 5.4 316.1 21.5 549.9 22.1 80.7 6.1 33.0 140 104.3 8.1 57.6 2 86.6 5.3 272.8 22.6 467.9 24.8 78.0 5.6 25.0 150 127.9 11.2 41.5 3 73.7 7.2 332.8 12.9 418.4 22.3 53.2 4.9 20.0 170 131.5 8.6 54.3 4 68.4 6.7 346.9 16.2 350.7 18.6 85.1 6.3 16.0 150 150.9 13.2 47.8 5 75.6 8.1 338.6 20.4 344.7 19.8 85.6 5.6 28.0 200 126.0 10.9 49.5 6 111 .4 8.2 337.6 17.0 358.5 20.3 80.0 5.5 27.0 210 105.9 12.9 43.1 7 94.7 5.6 329.8 23.4 413.5 16.5 69.6 4.6 34.0 170 113.2 9.0 53.2 8 83.3 6.6 297.2 22.7 792.3 28.6 80.3 7.3 14.0 175 125.8 7.7 33.6 9 63.9 5.1 375.0 21.3 335.2 33.0 93.8 5.7 13.0 114 105.7 11.7 58.8 10 90.9 6.1 290.9 17.7 400.3 23.4 74.2 6.2 20.0 139 121 .6 10.3 63.1 11 95.6 3.6 214.6 14.8 403.0 20.2 83.2 5.7 20.0 111 122.1 7.8 43.8 12 106.7 5.8 329.6 9.5 449.0 23.4 74.9 5.0 25.0 111 110.0 9.6 77.5 13 99.6 7.3 173.7 17.2 438.8 27.9 85.0 6.0 15.0 90 130.2 7.6 49.1 14 88.2 5.3 286.1 23.8 370.3 30.3 60.5 4.9 19.0 71 151.2 12.4 43.6 15 70.2 4.5 332.7 23.9 468.0 29.0 66.8 5.1 19.5 96 120.7 9.1 54.6 16 97.7 6.8 346.0 13.8 427.3 10.0 67.6 5.7 17.0 100 124.8 10.3 63.9 17 67.1 6.8 253.4 18.2 441 .8 19.8 78.7 4.9 21.0 87 131 .7 7.1 49.2 18 80.6 5.9 274.8 20.3 480.4 24.6 93.3 4.9 23.0 135 103.5 10.7 54.4 19 85.1 5.3 349.3 17.3 509.1 26.9 77.9 5.4 13.0 91 135.3 9.1 54.8 20 63.8 6.8 332.8 22.7 403.7 16.1 75.3 8.0 16.0 97 137.5 12.0 62.5 21 71 .8 8.9 308.2 11.8 404.2 26.3 65.8 5.3 129.5 8.0 56.0 22 87.9 8.2 329.3 24.8 384.0 24.5 77.2 6.0 108.8 8.0 69.6 23 4.9 330.9 18.1 482.9 23.4 79.0 4.6 108.8 9.6 62.9 24 8.1 314.5 20.4 438.5 20.5 85.1 3.7 133.7 6.4 54.5 25 6.2 298.6 19.3 428.2 26.7 98.1 4.4 120.0 9.3 75.5 26 341 .5 23.2 458.8 19.2 75.1 5.8 101.5 7.0 63.3 27 328.0 22.6 510.9 24.3 67.5 4.1 98.9 12.3 54.7 28 203.0 11 .9 483.8 32.0 82.3 5.4 119.2 7.0 69.9 29 343.5 18.8 513.6 21.1 69.3 5.3 116.3 9.1 58.9 30 383.0 20.5 526.3 16.0 73.9 5.8 82.1 10.3 44.9 31 337.8 21.3 399.0 33.9 73.8 5.7 121 .8 9.7 67.3 32 285.9 23.0 349.3 22.2 92.8 5.6 123.2 8.1 63.9 33 320.6 13.3 466.1 20.5 90.3 6.9 139.5 7.6 51 .3 34 292.1 6.2 428.8 17.2 85.0 6.7 111 .0 8.8 58.4 35 269.1 22.3 381.0 16.6 79.0 7.0 127.4 9.1 63.2 36 353.6 20.7 529.4 17.3 69.0 6.5 116.2 5.3 57.4 37 292.4 12.8 477.8 15.3 76.9 4.9 140.2 8.1 53.2 38 185.1 21 .5 454.8 36.2 87.7 6.4 132.9 10.0 55.1 39 248.0 18.1 407.5 18.2 79.6 5.5 113.5 7.4 63.5 40 284.7 18.1 516.1 24.8 73.3 6.5 84.1 7.3 59.7 41 310.2 23.4 448.7 25.7 79.8 5.2 100.2 9.8 54.7 42 314.9 26.3 438.5 23.2 82.4 4.4 125.2 9.6 52.3 43 274.7 17.6 447.3 17.2 83.8 4.4 132.9 6.6 55.8 44 301.2 18.4 546.4 18.2 73.1 3.9 122.8 13.0 62.2 45 371.9 25.8 540.5 19.3 91 .5 3.9 122.9 9.2 65.3 46 297.7 19.6 441 .6 29.2 84.8 4.8 139.5 10.0 56.4 47 290.3 17.5 447.8 22.5 61.4 5.4 135.2 9.7 37.5 48 264.1 14.9 371 .9 22.4 92.2 5.2 118.4 9.6 63.9 49 363.7 17.4 479.4 28.8 78.4 5.7 106.8 4.3 58.8 50 236.0 18.4 448.1 23.5 89.3 5.5 127.4 9.9 59.7 70

APPENDIXD

MEASUREMENTS OF SPICULES FOUND IN DOCOSACCUS MACULATUS 71

Gastral pentactin Gastral hexactin Dermal hexactin Tangential Tangential Proximal Proximal Ray Tangential Tangential Proximal Proximal Distal ray Distal ray Ray Width ray length ray width ray length ray width length ray length ray width ray length ray width length width 1 329.0 25.7 500.1 19.0 285.8 11.3 391 .5 16.5 659.3 19.8 66.2 22.4 2 241.3 26.1 431.4 15.2 292.5 11.6 279.1 18.8 638.6 25.8 139.1 21.3 3 350.2 21.3 399.2 27.0 237.2 8.7 348.9 17.1 396.8 25.3 219.7 17.5 4 335.0 31.0 476.3 13.4 302.5 12.6 275.3 17.2 813.2 17.3 134.5 20.6 5 307.2 29.0 399.3 16.3 301.9 12.9 405.1 16.0 592.7 22.7 151.5 18.2 6 387.7 36.6 417.3 19.0 323.4 29.6 366.6 28.0 466.7 16.6 120.9 20.7 7 311 .9 29.3 593.1 15.7 357.6 8.8 277.8 13.9 446.2 10.7 188.7 18.3 8 316.8 35.8 355.3 15.2 299.6 6.3 414.4 13.0 487.5 20.6 189.9 19.6 9 401.0 29.1 491.3 15.2 264.3 8.2 466.8 26.6 509.1 22.9 190.6 19.4 10 275.4 22.4 583.3 12.0 270.8 15.6 318.0 17.4 551 .7 17.1 133.7 20.8 11 319.3 24.0 355.9 11.4 205.3 14.6 419.2 22.4 605.8 22.2 122.7 18.7 12 391 .3 22.7 404.9 12.0 356.5 4.3 379.9 31 .1 1134.9 22.5 121.4 28.1 13 304.8 31.0 397.7 10.7 357.3 15.1 397.5 20.4 810.7 22.7 145.4 25.2 14 353.1 31.9 604.0 17.0 293.6 8.4 354.3 16.9 445.7 13.7 98.7 15.3 15 314.4 26.1 457.7 19.6 277.1 6.5 260.3 29.4 673.2 20.6 125.3 21.6 16 291.4 30.5 402.9 19.0 334.1 4.2 402.8 10.8 427.0 26.0 102.0 16.8 17 538.5 16.1 290.5 6.2 259.4 24.0 375.5 17.6 198.3 32.4 18 469.8 20.5 328.9 8.8 381.8 26.0 626.3 23.3 227.7 13.1 19 473.1 17.0 270.4 2.8 293.6 22.8 910.6 13.2 165.0 12.2 20 490.3 13.4 501 .1 13.6 195.0 24.8 780.3 31.4 157.5 22.0 21 543.0 20.5 339.7 11.0 381.8 17.7 726.7 21.4 130.3 16.2 22 538.1 20.5 331.0 5.9 375.1 17.8 692.6 24.7 100.2 15.3 23 604.9 16.1 289.0 10.7 281 .8 13.8 435.4 27.7 161.1 24.8 24 672.4 28.0 348.3 14.8 436.3 26.3 529.3 37.3 151.6 15.7 25 499.6 16.1 430.2 8.3 240.8 24.0 441.6 15.5 115.4 23.6 26 337.2 21 .0 417.5 9.7 302.1 20.7 710.4 15.5 117.5 21.7 27 373.4 19.6 350.2 4.0 300.0 23.5 445.5 21.8 107.5 12.9 28 472.7 16.1 321 .2 3.2 27.4 721 .0 25.0 232.8 22.0 29 619.0 17.2 258.4 5.4 16.7 780.8 33.6 114.3 24.2 30 432.5 13.4 301 .3 6.6 20.1 356.5 17.3 116.9 20.7 31 550.3 13.7 275.1 3.7 31 .6 400.8 11.8 149.9 15.9 32 470.6 18.8 348.5 4.9 22.7 393.6 14.4 153.8 28.7 33 617.4 21.5 202. 1 5.2 16.5 408.1 16.7 146.4 15.8 34 574.0 16.1 305.9 10.1 26.9 837.4 26.4 146.1 13.0 35 456.2 18.0 289.6 10.7 591.4 24.4 98.4 13.6 36 457.0 17.2 325.6 8.9 516.4 25.5 170.4 16.4 37 529.7 10.7 315.4 13.5 412.2 21 .6 220.2 13.7 38 410.5 13.4 390.2 17.2 544.8 28.6 125.2 16.6 39 535.1 17.2 374.7 16.9 444.2 25.4 112.6 16.6 40 358.4 13.7 277.3 22.6 568.5 26.7 102.0 19.5 41 392.8 18.0 324.8 27.8 484.8 19.7 119.1 24.9 42 417.4 15.7 241 .6 19.4 686.4 24.5 128.6 21.5 43 509.6 20.5 413.4 26.7 582.4 27.3 121.0 28.9 44 1332.4 14.5 236.7 23.7 595.7 19.1 107.4 29.9 45 501.7 19.4 279.5 19.0 731.1 27.6 100.3 18.6 46 452.8 14.5 275.0 22.5 594.5 30.7 113.2 10.4 47 615.2 19.4 349.7 20.9 733.7 17.2 152.5 29.9 48 798.2 22.1 293.1 18.5 733.4 23.5 113.9 23.3 49 483.7 14.5 294.0 26.5 777.7 30.7 225.9 25.1 50 356.3 26.9 279.1 31 .5 141.1 19.3 72

Giant hexactin (mm) Oxyhemihexaster Floricome

Tangential Tangential Diameter Primary Secondary Diameter Primary Secondary rays (long) and basal ray ray length ray length ray length ray length 1 30 6 44.7 6.4 34.4 68.8 10.0 42.2 2 27 5 42.7 5.3 28.5 56.9 9.5 48.4 3 21 4 34.9 7.9 35.8 71 .5 7.1 47.7 4 22 14 41 .2 8.8 37.5 75.1 9.1 44.2 5 36.4 6.9 31.1 62.1 9.7 40.9 6 41.5 8.1 33.1 66.1 7.5 38.0 7 36.6 5.9 34.6 69.2 9.6 48.0 8 39.2 7.4 36.0 71 .9 7.3 32.8 9 38.8 7.7 28.3 56.5 9.6 45.6 10 41 .6 7.4 34.6 69.1 7.3 41 .8 11 37.4 9.3 29.1 58.2 8.3 42.2 12 56.1 7.4 32.0 63.9 5.2 32.5 13 50.4 8.7 24.3 48.6 7.8 43.3 14 30.6 6.4 25.5 51 .0 11 .9 51.3 15 43.2 6.2 26.1 52.1 8.1 28.4 16 33.5 8.6 33.9 67.9 7.9 51.1 17 64.8 6.2 33.1 66.2 7.1 44.7 18 26.2 7.1 36.3 72.6 7.5 51 .8 19 24.3 4.9 31 .1 62.2 7.5 51.6 20 44.1 6.7 27.4 54.7 9.5 43.7 21 32.4 7.6 41 .7 83.5 5.7 50.6 22 30.7 9.1 34.7 69.3 10.5 44.1 23 49.6 7.2 32.1 64.2 24 31.4 4.1 31 .5 63.0 25 47.1 8.9 30.5 60.9 26 43.4 7.9 34.2 68.5 27 25.7 8.7 36.2 72.4 28 44.0 9.1 31.3 62.5 29 48.3 8.3 28.8 57.5 30 41.4 7.9 32.8 65.6 31 31.7 8.0 32.1 64.3 32 57.3 9.1 31.1 62.3 33 31.6 7.9 25.6 51 .2 34 26.1 8.7 32.6 65.3 35 27.3 8.6 30.5 61 .1 36 32.8 8.3 29.4 58.7 37 27.4 6.7 26.7 53.4 38 33.2 5.9 28.2 56.5 39 33.1 7.5 27.6 55.2 40 39.0 8.1 34.1 68.1 41 49.7 5.7 36.8 73.7 42 43.1 8.5 30.3 60.6 43 57.9 8.0 31.9 63.7 44 59.8 4.3 35.9 71 .8 45 37.2 10.1 35.2 70.4 46 20.8 6.4 36.5 73.0 47 59.7 8.3 33.3 66.6 48 46.6 7.7 37.1 74.3 49 50.3 8.3 28.8 57.7 50 38.7 6.8 26.1 52.3