Meio-epifauna of Costa Rican Cold Seeps

María Adriana Gracia Clavijo

Universidad Nacional de Colombia Facultad de Ciencias, Departamento de Biología Instituto de Estudios en Ciencias del Mar – CECIMAR Santa Marta D.T.C.H., Colombia 2018

Meio-epifauna of Costa Rican Cold Seeps

María Adriana Gracia Clavijo

Tesis de investigación presentada como requisito parcial para optar al título de: Doctor en Ciencias - Biología

Director: Sven Zea, Ph.D. Codirectora: Lisa A. Levin, Ph.D.

Línea de Investigación: Biología Marina

Universidad Nacional de Colombia Facultad de Ciencias, Departamento de Biología Instituto de Estudios en Ciencias del Mar – CECIMAR Santa Marta D.T.C.H., Colombia 2018

A mi madre……… A Nelson y la pequeña gran familia que formamos…

Acknowledgements

In a very special way, I want to thank my two thesis directors Professors, Dr. Sven Zea, and Dr. Lisa Levin. Thanks to both of them for their dedication and effort in this thesis. I appreciate receiving from two of the greatest marine researchers all their knowledge and guidance always to be better.

Several people have contributed to this study through their participation. To Dr. Javier Sellanes (Universidad de Concepción), and the professors Dr. Arturo Acero and Dr. Nestor Campos (Instituto de Estudios en Ciencias del Mar – CECIMAR, Universidad Nacional de Colombia, Caribbean Campus) for their support in the first part of the elaboration of my thesis project. To Ana Milena Cardenas (Universidad Nacional de Colombia) for her excellent collaboration in all the administrative processes of the University. This doctoral dissertation was supported by Colciencias- Colfuturo National Scholarship Grant N° 528.

Thanks to Dr. Lisa Levin for let me be a part of her research developed at Scripps Institution of Oceanography - UC San Diego. Also, to Dr. Ben Grupe, who was one of the leaders of the macro project in which this thesis was involved. To Jennifer Gonzales for all her support and collaboration in the laboratory and sample management. Also, to all personnel involved in the cruises conducted by Scripps Institution. The research was supported by the National Science Foundation (NSF grants OCE 0826254 and OCE 0939557).

To Dr. Gustavo Fonseca and colleagues for their support during the internship in nematodes carried out in his laboratory (Instituto do Mar - UNIFESP, Santos, Brazil). To Emeritus Professor Dr. William Neal (Grand Valley State University) for his contributions and improvements to Chapter 3. To José Manuel Gonzales (Universidad Nacional de Colombia) for his friendship, and all his guidance and support in the management of information, specifically in Chapter 4. To Fernando Dorado (Invemar) for his support, comments, and improvements to Chapter 4. To Dr. Carlos Neira (Scripps Institution) for his comments and improvements to Chapter 5. To Nadia Santodomingo for her friendship and good advice. To my colleagues at Invemar for their support and good timeshare at the Museum. Especially to Andrea Polanco, Erika Montoya and Miguel Martelo.

I would like to thank Nelson Guillermo and his infinite effort for understanding some of what I've done over the years. Everything has been words of encouragement and support.

Abstract & Resumen IX

Abstract

Reducing marine environments include hydrothermal vents, cold seeps, whale carcasses, oxygen minimum zones, sunken wood, accumulated loose seagrasses and other organic remains. Cold seeps are ocean floor areas where pore waters rich in CH4 and H2S, wich reach the upper sediment layer; Temperatures are usually at ambient deep-sea values. Macrofaunal and megafaunal species exhibit symbiotic associations with methane-oxidizing and sulfur-oxidizing bacteria. In this environment, there also are meiofaunal communities (> 42 µm) which throphically link bacterial and macrofaunal food webs. Although ubiquitous and more abundant compared to larger-sized invertebrates, meiofaunal communities are among the least studied and understood components of the deep-sea benthos. The best known meiofaunal community dwells within sea-floor sediments, but an important community, called meio-epifauna, inhabits solid surfaces (i.e., rocks, wood, biogenic material) usually present in these reducing environments, is almost unknown. The presence of these substrates can influence diversity at the microscale through the provision of habitat, food, shelter, and various biotic interactions. In the eastern Pacific, the Costa Rican continental margin has heterogeneous areas with different combinations of reducing systems. This work at seep areas of Costa Rica's Pacific Ocean (between ~380 and 1800 m deep) employed natural substrate samples and in situ experiments (for 10.5 months) to investigate at several scales the relationships between locations, seepage activity, habitats, and substrates with taxon richness, abundance, and community structure of meio-epifaunal metazoans, and the controls over their presence and functions. This dissertation covers a broad range of colonization topics. The first chapter provides an introduction to the study area including some meiofaunal biological aspects. The second chapter looks for understanding on a large scale (six locations) meio-epifauna colonization processes associated with inorganic substrates (authigenic rocks) deployed at actively seeping and inactive sites. The analysis was carried out on both natural and experimental rocks. A rich community was found consisting of 27 meio- epifaunal taxa in natural rocks. Nematodes (58.1 %) followed by copepods (24.9 %) were the most abundant. Inactive areas supported for the highest mean number of individuals (19.2±17.6 (1 SD) ind.10cm-2, n=17). Significant differences in higher taxa composition and abundance were found between locations (Jaco scar, Jaco wall, Mound 11, Mound 12, Mound Quepos, Quepos landslide) and between habitats (Bacterial mats, carbonates, hard coral, mussel beds, near clam beds, Oxygen Minimum Zone, sediments, tubeworms), but not between seepage activities. Different locations exhibit different hydrographic characteristics, with depth and temperature being the variables that best explain the biological scheme found. After 10.5 months of colonization of authigenic rocks, the community was comprised of 14 meio-epifaunal taxa. Copepods accounted for the highest abundances (69 %), followed by nematodes (18 %). Also, the highest mean density was found in inactive areas (26.3±11.3 ind. 10cm-2, n=5) compared to active areas (16.4±16.4 ind. 10cm-2, n=6). As in natural rocks, in Mound 12 carbonate colonizers, significant differences were found between habitats but not between seepage activities. Nematodes were thus confirmed to be the dominant

meio-epifaunal group inhabitants of deep-sea environments, but our colonization experiments suggest that copepods are better represented in the early stages of colonization. Chapter 3 discussed colonization on a smaller scale (Mound 12) using organic substrates (wood blocks). In this case, a less diverse community was found with nine meio-epifaunal higher taxa and a total of 9,951 individuals. Copepods accounted for the highest abundances (75 %), followed by nauplii larvae (12 %), and nematodes (10 %). The maximum number of individuals (26.3 ind.10cm-2) was found in wood blocks placed in inactive areas (near active mussel beds). Analysis grouped blocks according to seepage activity and not to habitat, but tests of similarity showed no significant differences in higher taxon composition and abundances, probably owing either to substrate homogeneity or low sample size. Contrary to previous studies, where nematodes appeared as the dominant colonizers in deep-sea environments, in the wood blocks used, copepods were the most abundant representatives, suggesting that this group is one of the most successful colonizing hard-wood substrates that are not yet decomposed. Chapter 4 dealt with copepods in experimental substrates (i.e., wood blocks, carbonates rocks, polychaetes tubes, and clam shells). A total of 24,467 individuals belonging to five orders and 15 families were identified. The order Harpacticoida was the best-represented with respect to families (7) and density (87.5 %). Miraciidae (38.6 %), Ectinosomatidae (34.2 %), Tegastidae (9.4 %) and Ameiridae (4.9 %), accounted for approximately 87.1 % of all copepods density. The highest densities of copepods occur in inactive areas, regardless of the type of experimental substrate, had the highest densities of copepods (16.4±9.9 (1 SD) ind.10cm-2, n=9) compared to active areas (8.8±9.9 ind.10cm-2, n=11). The cluster analysis revealed the presence of a typical configuration (stepped) of assemblages, related to environmental gradients. At the family level analyzed, the assemblage responds to characteristics given by the seepage activity. Also, a turnover in family dominance between Miraciidae and Ectinosomatidae according to seepage activity was observed. Lastly, Chapter 5 used nematodes to examine trophic structure and habitat relationships on natural rocks at Mounds 11 and 12. Nematode faunas were represented by two classes, six orders, 17 families, and 27 genera. The family Xyalidae was most abundant (30.1 %) followed by Comesomatidae (21.2 %) and Linhomoeidae (20.9 %). Daptonema and Metalinhomoeus were the genera reaching the highest density (29.3 % and 18.2 % respectively) in all surveys. The results demonstrate the existence of slight differences between habitats regarding nematode assemblage structure. On the substrates, the assemblages were composed of nematode taxa with different trophic features. Non-selective deposit-feeders were dominant (58 %) followed by epigrowth feeders (29 %). At the family and genus level, the nematode fauna was found to be similar to that of other deep-sea locations of cold seep areas.

Keywords: Methane seep, Meiofauna, Deep-sea colonization, Hard substrates, Costa Rica margin, Eastern Pacific, Copepoda, Nematoda, Carbonate, Wood.

Abstract & Resumen XI

Resumen

Los ambientes reductores marinos incluyen respiraderos hidrotermales, filtraciones frías, carcasas de ballenas, zonas de oxígeno mínimo, madera hundida, cumulos sueltos de pastos marinos y otros restos orgánicos. Las filtraciones frías son áreas del fondo oceánico donde aguas ricas en CH4 y H2S llegan a la capa sedimentaria superior; Las temperaturas suelen estar en los valores ambientales de las profundidades marinas. Una amplia gama de especies macro y megafaunales exhiben asociaciones simbióticas con bacterias oxidantes del metano y del azufre. En este entorno, también existen comunidades meiofaunales (> 42 µm) las cuales se caracterizan por enlazar las redes alimentarias bacterianas y macrofaunales. Aunque son ubicuas y más abundantes que los invertebrados de mayor tamaño, las comunidades meiofaunales son uno de los componentes menos estudiados y comprendidos del bentos profundo. Los organismos que componen la meiofauna generalmente viven dentro de los sedimentos del fondo marino, pero la otra comunidad importante llamada meio-epifauna, que habita superficies sólidas (i.e., rocas, maderas, material biogénico) usualmente presentes en estos ambientes reductores es casi desconocida. La presencia de estos sustratos puede influir en la diversidad a microescala a través de la provisión de sustrato, alimento, refugio y varias interacciones bióticas. En el Pacífico oriental, el margen continental costarricense presenta áreas heterogéneas con diferentes combinaciones de sistemas reductores. Este trabajo en áreas de filtraciones frías del océano Pacífico de Costa Rica (entre ~380 y 1800 m de profundidad) utilizó muestreos de rocas naturales y experimentos in situ (de 10.5 meses de duración) para investigar a diferentes escalas las relaciones entre localidades, presencia de actividad (filtración) reductora, hábitats, y sustratos, con la riqueza taxonómica, abundancia y estructura comunitaria de la meio-epifauna, y los controles sobre su presencia y funciones. Esta disertación cubre una amplia gama de temas sobre colonización. El primer capítulo ofrece una introducción al área de estudio, incluyendo algunos aspectos biológicos sobre la meiofauna. El segundo capítulo busca comprender a gran escala (seis localidades) los procesos de colonización de la meio-epifauna asociada a sustratos inorgánicos (rocas autigénicas) localizados en sitios tanto activos como inactivos. El análisis se llevó a cabo tanto con rocas naturales como experimentales. Se encontró una rica comunidad compuesta por 27 taxones meio-epifaunales en rocas naturales. Los nematodos (58,1 %) seguidos por los copépodos (24,9 %) fueron los más abundantes. En las áreas inactivas se encontraron el mayor número medio de individuos (19,21± 17,63 (1 SD) ind.10cm-2, n= 17). Se encontraron diferencias significativas entre las localidades (Jaco scar, Jaco wall, Mound 11, Mound 12, Mound Quepos, Quepos landslide) y entre los hábitats (Tapetes de bacterias, carbonatos, coral duro, bancos de mejillones, cerca de bancos de almejas, zona de mínimo oxígeno, sedimentos, tubos de poliquetos), pero no entre la actividad de la filtración. Las diferentes localidades presentaron diferentes características hidrográficas, siendo la profundidad y la temperatura las variables que mejor explican el esquema biológico encontrado. Después de 10,5 meses de colonización, la comunidad estaba compuesta por 14 taxones meio-epifaunales. Los copépodos representaron las mayores abundancias

(69 %), seguidos por los nematodos (18 %). También se encontró la mayor densidad media en áreas inactivas (26,3± 11,3 ind. 10cm-2, n= 5) comparado con áreas activas (16.4±16.4 ind. 10cm-2, n=6). Al igual que en las rocas naturales, en los colonizadores del Montículo 12 se encontraron diferencias significativas entre hábitats pero no entre actividades. Por lo tanto, se confirmó que los nematodos son el grupo meio-epifaunal dominante de los ambientes de aguas profundas, pero los experimentos de colonización sugieren que los copépodos están mejor representados en las primeras etapas de la colonización. El capítulo tres examinó la colonización a menor escala (Montículo 12) utilizando sustratos orgánicos (bloques de madera). En este caso se encontró una comunidad menos diversa con nueve taxones meio-epifaunales, y un total de 9.951 individuos. Los copépodos representaron las mayores abundancias (75 %), seguidos por las larvas de nauplios (12 %) y los nematodos (10 %). El número máximo de individuos (26,3 ind.10cm-2) se encontró en bloques de madera colocados en áreas inactivas (cerca de los bancos de mejillones, que se ubican en áreas activas). El análisis agrupó los bloques según la actividad de la filtración y no según el hábitat, pero las pruebas de similitud no mostraron diferencias significativas en la composición y abundancia de los taxones, probablemente debido a la homogeneidad del sustrato o al bajo tamaño muestreal. Contrariamente a estudios anteriores, donde los nematodos aparecieron como colonizadores dominantes en ambientes de aguas profundas, en los bloques de madera usados, los copépodos fueron más abundantes, sugiriendo que este grupo es uno de los más exitosos en colonizar en la etapa temprana de sucesión sobre sustratos de madera que no han comenzado a descomponerse. El capítulo cuatro se ocupó de los copépodos en sustratos experimentales (es decir, bloques de madera, rocas carbonatadas, tubos de poliquetos y conchas de almejas). Se identificaron un total de 24.467 individuos pertenecientes a cinco órdenes y 15 familias. El orden Harpacticoida fue el mejor representado en cuanto a familias (7) y densidades (87,5 %). Miraciidae (38,6 %), Ectinosomatidae (34,2 %), Tegastidae (9,4 %) y Ameiridae (4,9 %), representaron aproximadamente el 87,1 % de la densidad total de copépodos. En áreas inactivas, independientemente del tipo de sustrato experimental, se presentaron las mayores densidades de copépodos (16,4± 9.9 (1 SD) ind.10cm-2, n=9) comparado con áreas activas (8.8±9.9 ind.10cm-2, n=11). Los análisis multivariados mostraron una configuración típica de los ensamblajes en escalera, que se relacionan con grandientes ambientales. A nivel de familia, el ensamblaje responde a características dadas por la actividad de la filtración. Además, se observó un cambio en el predominio entre las familias Miraciidae y Ectinosomatidae según la actividad de la filtración. Por último, el capítulo cinco utiliza los nematodos para examinar la estructura trófica y las relaciones del hábitat en las rocas naturales de los montículos 11 y 12. La fauna de nematodos estuvo representada por dos clases, seis órdenes, 17 familias y 27 géneros. La familia Xyalidae fue la más abundante (30,1 %) seguida por Comesomatidae (21,2 %) y Linhomoeidae (20,9 %). Daptonema y Metalinhomoeus fueron los taxones con mayor densidad acumulada (29,3 % y 18,2 % respectivamente) en todo el estudio. Los resultados demuestran la existencia de ligeras diferencias entre hábitats en cuanto a la estructura de ensamblaje de nematodos. En los sustratos, los ensambles estaban compuestos por taxa de nematodos con diferentes características tróficas. Los alimentadores de depósito no selectivos fueron los dominantes (58 %) seguidos por los alimentadores de superficie (29 %). A nivel de familia y género, se encontró que la fauna de nematodos era similar a la encontrada en otros lugares de filtraciones frías de aguas profundas. Palabras clave: Filtraciones de metano, Meiofauna, Colonización de aguas profundas, Sustratos duros, Margen de Costa Rica, Pacifico oriental, Copepoda, Nematoda, Carbonatos, Maderas. Contents XIII

Contents

1. Geological and biological background ...... 5 1.1 Geological background ...... 5 1.2 Meiofauna ...... 6 1.3 Ecological relevance of colonization ...... 7 1.4 Habitats and substrates ...... 8 1.5 General methodology ...... 9 1.5.1 Field methods ...... 9

2. Meio-epifaunal colonization on carbonate substrates in deep cold seeps of the Costa Rican Pacific margin ...... 11 2.1 Introduction ...... 12 2.2 Material and Methods ...... 15 2.2.1 Study area ...... 15 2.2.2 Habitat sampling ...... 18 2.2.3 Density quantification ...... 18 2.2.4 Data analyses ...... 19 2.3 Results ...... 20 2.3.1 Composition and abundance of natural rocks ...... 20 2.3.2 Community patterns of natural rocks ...... 23 2.3.3 Abundance and composition of experimentally deployed substrates...... 30 2.4 Discussion ...... 34

3. Meio-epifaunal wood colonization in the vicinity of methane seeps ...... 49 3.1 Introduction ...... 50 3.2 Material and Methods ...... 52 3.2.1 Costa Rica Marine Setting and Mound 12 ...... 52 3.2.2 Study area ...... 52 3.2.3 Habitats selection ...... 53 3.2.4 Colonization experiments on Mound 12 ...... 53 3.3 Results ...... 56 3.3.1 Wood block description ...... 56 3.3.2 Meio-epifaunal composition ...... 56 3.3.3 Community analysis ...... 59 3.3.4 Wood associated meiofauna and macrofauna on reducing deep-sea environments ..... 61 3.4 Discussion ...... 62

4. Meio-epifaunal copepods (Crustacea) assemblages in experimental susbtrates at Mound 12 cold seep ...... 73 4.1 Introduction ...... 74 4.2 Methods ...... 76 4.2.1 Study area ...... 76 XIV Meio-epifauna Costa Rica

4.2.2 Habitat sampling ...... 78 4.2.3 Colonization experiments on Mound 12 ...... 78 4.2.4 Density quantification ...... 79 4.2.5 Data analyses ...... 81 4.3 Results ...... 82 4.3.1 Faunistic variables...... 82 4.3.2 Copepod spatial community structure ...... 94 4.4 Discussion ...... 98 4.4.1 Faunistic composition ...... 98 4.4.2 Community and assemblage’s analysis ...... 100

5. Trends in trophic structure and habitat preferences in authigenic cold seep rock meio- epifaunal nematodes at Mounds 11 and 12 of Costa Rica ...... 103 5.1 Introduction ...... 103 5.2 Methods ...... 106 5.2.1 Study area ...... 106 5.2.2 Collection of natural rocks ...... 107 5.2.3 Quantification of abundance ...... 107 5.2.4 Data analyses ...... 108 5.2.5 Nematode trophic structure ...... 108 5.1 Results ...... 111 5.1.1 Abundance and nematode taxonomic diversity ...... 111 5.1.2 Community patterns ...... 117 5.1.3 Nematode trophic structure ...... 121 5.2 Discussion ...... 123

6. Conclusions and recomendations ...... 129 6.1 Conclusions ...... 129 6.2 Recomendations ...... 132

7. References ...... 133

Contents XV

List of figures

Pág. Figure 2-1: A. Study locations on cold seeps on the Costa Rica continental margin in the Pacific Ocean. B-E. Photos of some substrate samples taken on board immediately after retrieval (with permission from Greg Rouse), B. Jaco wall, C. Mound 11, D. Mound 12, E. Mound Quepos. Cruise AT-15-44. For scale see the hand holding the rocks...... 16 Figure 2-2: A. Habitats where experimental rock substrates were deployed on Mound 12 for 10.5 months of colonization on Cruise AT-15-44, with recovery on AT 15-59. B-D. Photos of some experimental rocks after retrieval (numbers indicate diving logs; pictures with permission from Greg Rouse). A=Active, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, RB=Rubble, SED=Sediments, TW=Tubeworms...... 17 Figure 2-3: Relative abundance (% of total density) of different taxonomic groups of meio- epifauna found in natural rocks as function of habitat (marked at the top of the bars). A=Active, BM=Bacterial mat, BoulF=Boulder Field, Carb=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco summit, JW=Jaco wall, MB=Mussel beds, MQ=Mound Quepos, M11=Mound 11, M12=Mound 12, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, TW=Tubeworms. n=number of substrates per location...... 21 Figure 2-4: A. Contribution (% ind. 10 cm-2) of different groups to the total meio-epifauna found in each location on natural rocks. B. Mean (±SD) number of taxa found in each location...... 22 Figure 2-5: Contribution (% ind. 10 cm-2) of different groups to the total meio-epifauna found in each habitat on natural rocks. A=Active, BM=Bacterial mat, Carb=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, TW=Tubeworms...... 23 Figure 2-6: A. Cluster analysis and B. two-dimensional MDS ordination of meio-epifauna associated with natural rocks of Costa Rica cold seeps. In the dendrogram (A), the four clusters, produced by a threshold similarity of 65 % are shown, however SIMPROF test support five (A to E) main groups (black lines). In the ordination (B) green lines show four distinct groups (D nested within E) separated by a threshold similarity of 65 %. A=Active, BM=Bacterial mats, BoulF=Boulder Field, Car=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco scar, JW= Jaco wall, M11=Mound 11, M12=Mound 12, MB=Mussel beds, MQ=Mound Quepos, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, Sed=Sediments, TW=Tubeworms...... 24 Figure 2-7: Relative abundance of meio-epifauna in experimental rocks after 10.5 months of colonization (ordered by habitat and seepage activity). Densities are averaged by habitat. Labels show the number (#) of each rock deployed in each habitat-activity combination; there was only one rock (R#11) in the sediment habitat. A=Active, I=Inactive, NMB=Near mussel bed, RB=Rubble, Sed=Sediments, TW=Tubeworms...... 31 Figure 2-8: Mean density (ind.10 cm-2±1 SD) in experimental rocks after 10.5 month of colonization (Mound 12), all rocks deployed in inactive sites (near mussel bed, in rubble and in XVI Meio-epifauna Costa Rica sediments) combined, in comparison to active sites. A=Active, MB=Mussel bed, NMB=Near mussel bed, TW= Tubeworms...... 31 Figure 2-9: A. Cluster analysis and B. two-dimensional MDS ordination of meio-epifauna associated with 11 experimental rocks of Costa Rica cold seeps after 10.5 months of colonization. In A., there is a single homogeneous group (red lines) from SIMPROF (the black broken line marks 65 % similarity level). In B., green lines group clusters produced in A. at the 65 % similarity threshold. A=Active, I=Inactive, NMB=Near mussel bed, RB=Rubble, Sed=Sediments, TW=Tubeworms. R#=sample number...... 33 Figure 2-10: Mean density ±1 SD (ind.10 cm-2) comparing main taxa between natural (blue) substrates and in experimental rocks (red) after 10.5 months of colonization. Others summarize all other taxa found different from the most representative groups...... 34 Figure 3-1: Map of the study area showing the location were wood blocks were deployed in Mound 12, Costa Rica. A-MB (active, mussel bed)=location of Woods #1 and #4; I-NMB (inactive, near mussel bed)=Wood #2; A-TW (active, tubeworms)=Woods #5 and #6; I-RB (inactive, rubble bottom)=Woods #7 and #8. Each wood block is illustrated just at recovery (Wood #3, located in an inactive site near mussel bed habitat was lost). Wood pictures by permission from Greg Rouse, Scripps Institution of Oceanography...... 55 Figure 3-2: Relative abundance (% of total density) of different taxa of meio-epifauna found on each wood block. Abbreviations of each block are Wood number (W#), seepage activity (A=Active, I=Inactive), and habitat (MB=Mussel bed, NMB=Near mussel bed, RB=Rubble, TW=Tubeworms)...... 57 Figure 3-3: Relative abundance (% of number of total meio-epifaunal density) per taxa combining all wood blocks by seepage activity...... 58 Figure 3-4: Relative abundance (% of total meio-epifaunal density) per taxa combining wood blocks for each habitat. MB=Mussel beds, NMB=Near mussel bed, RB=Rubble, TW=Tubeworms...... 58 Figure 3-5: Cluster (A) and MDS ordinations (B-C) of pairwise Bray-Curtis similarity coefficients of meio-epifaunal assemblages of wood blocks after 10.5 months of deployment. Data standardized to numbers of individuals per 10 cm2, and fourth root tranformed. (Red=Inactive sites, Blue=Active sites). The two groups produced by a threshold similarity of 70 % are shown. The group of blocks depicted by a continuous red line is homogeneous and significantly different from the other, from SIMPROF. (C) MDS zoom of the tightly grouped blocks, having the same pattern of active vs. inactive segregation found in the cluster analysis...... 61 Figure 4-1: A. Map of the study area is showing the location where substrates were deployed in Mound 12 Costa Rica. B-C. Pictures taken in situ by U. San Diego, USA, during the recovery of experimental substrates: B. Authigenic carbonates and wood blocks; C. Lamellibrach tubes. Background grey regions indicate areas of carbonate cover, orange regions indicate bacterial mats, yellow regions indicated mussel beds or tubeworms clumps. CR=indicate the station number that group together different substrates and habitats combinations. This is used to follow the nomenclature assigned by Grupe (2014). Map based on Grupe (2014)...... 77 Figure 4-2: A. Relative abundance (%) by individual substrate of different copepod meio- epifaunal families in active and inactive sites, and habitats. The most representative families are illustrated: B. Miraciidae, C. Ectinosomatidae, and D. Tegastidae. A=Active, I=Inactive, Contents XVII

MB=Mussel beds, NMB=Near Mussel Beds, RB=Rubble, Sed=Sediments, TW=Tubeworms. Type of substrates: C=Clams, R=Rocks, T=Tubes, W=Woods...... 84 Figure 4-3: Relative abundance (%) of copepod stages found in the meio-epifauna colonizing different substrates in active and inactive habitats at Mound 12. A=Active, BM=Bacterial mats, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, Sed=Sediments, TW=Tubeworms. Type of substrates: C=Clams, R=Rocks, T=Tubes, W=Wood...... 87 Figure 4-4 Mean total density of copepod families found in the different habitats, ordered according to seepage activity. A=Active, I=Inactive, NMB= Near mussel beds, MB=Mussel beds, RB=Rubble, Sed=Sediments, TW=Tubeworms. ANOSIM: Global R=0.707, p=0.004...... 93 Figure 4-5: Cumulative density of copepod family composition comparing rock and wood experimental substrates by seepage activity...... 94 Figure 4-6: A. Cluster analysis and B. two-dimensional MDS ordination of pairwise Bray-Curtis similarity coefficients of meio-epifaunal copepod family assemblages colonizing for 10.5 months different activity-habitat combinations (=stations of Fig. 4-1). Data were standardized to numbers of individuals per 10 cm2, and square root transformed (means of all substrates in each station). A., there is a single homogeneous group (red lines) from SIMPROF; stations according the presence of carbonated ground are indicated. In B., green and blue lines group clusters produced in A. at the 55 and 75 % similarity threshold. A=Active, BM=Bacterial mats, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, Sed=Sediments, TW=Tubeworms...... 95 Figure 4-7: RWizard algorithm Shannon's diversity is represented in the colour gradient (yellow- red). Circles indicate the number of families. Also given Rarity Index, Simpson's dominance, Pielou Uniformity, and Taxonomic Uniformity...... 98 Figure 5-1: A. Location of Mounds 11 and 12 cold seeps off Costa Rica continental margin where authigenic rocks were collected. Most abuntant taxa are illustrated: B. Daptonema, C. Metalinhomoheus and D. Dorylaimopsis...... 106 Figure 5-2: Contribution (%) of nematode genera composition summed for activity-habitat combinations in Mounds 11 and 12 off Costa Rica. A=Active, Bmat=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms...... 111 Figure 5-3: A. Mean number (+1 SD) of of nematode taxa (genera) per seepage activity. B. Mean density of nematode per seepage activity. In both plots, data are combined for Mounds 11 and 12...... 114 Figure 5-4: Contribution (%) of nematode genera composition summed for activity in Mounds 11 and 12 off Costa Rica. A=Active, I=Inactive, M12=Mound 12, M11=Mound 11...... 115 Figure 5-5: A. Mean number (+1 SD) of nematode taxa (genera) per habitat. B. Mean density of nematodes per habitat. In both plots, data are combined for Mounds 11 and 12. M12=Mound 12, M11=Mound 11, A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms...... 116 Figure 5-6: A. Cluster analysis, and B. Two-dimensional MDS ordination of meio-epifaunal nematodes associated with natural rocks of Mounds 11 and 12 of Costa Rica cold seeps. The main groups produced by a threshold similarity of 47 % are shown. Black lines in the dendrograme indicate groups that are established, red lines showed a substructure from the clustering for which SIMPROF showed no statistical support. A=Active, BM=Bacterial mat, BoulF=Boulder Field, XVIII Meio-epifauna Costa Rica

Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms...... 118 Figure 5-7: Total proportional abundance of nematode feeding types at Mounds 11 and 12 of Costa Rica. 1A: selective deposit feeders, 1B: non-selective deposit-feeders, 2A: epistrate feeders, 2B: predators and/or omnivores...... 121 Figure 5-8: Nematode feeding contribution by density per seepage activity (active vs. inactive sites across locations and habitats). 1A: selective deposit feeders, 1B: non-selective deposit-feeders, 2A: epistrate feeders, 2B: predators and/or omnivores...... 121 Figure 5-9: Density (total ind.10 cm-2) of nematode feeding groups per habitat (across locations). A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms. 1A=Selective deposit feeders, 1B=Non-selective deposit-feeders, 2A=Epistrate feeders, 2B=Predators and/or omnivores...... 122

Contents XIX

List of tables

Pág. Table 2-1: Results of the various ANOSIM analyses comparing composition and density of meio-epifaunal higher taxa between location, seepage activity, and habitat, for both natural carbonate rock substrates, and experimental (10.5 month) colonization rocks. A=Active, BM=Bacterial mats, BoulF=Boulder Field, Car=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco scar, JW=Jaco wall, M11=Mound 11, M12=Mound 12, MB=Mussel beds, MQ=Mound Quepos, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, Sed=Sediments, TW=Tubeworms. *= significant...... 25 Table 2-2: Similarity results calculated by SIMPER (28 substrates). Taxa contributing most to the similarity between ANOSIM analysis (between location, and between habitat). Groups were ranked according to their average similarities (Av. sim.) and taxa according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given. Nematodes are the most important contributors to similarity in most groups. Groups MQ, NCB, BouldF, and HCor less than 2 substrates in group. A=Active, BM=Bacterial mats, BoulF=Boulder Field, Car=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco scar, JW=Jaco wall, M11=Mound 11, M12=Mound 12, MB=Mussel beds, MQ=Mound Quepos, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, Sed=Sediments, TW=Tubeworms...... 27 Table 2-3: BEST-BIOENV results. Only the best four correlations are shown. Best result for each number of variables are in bold. * best result...... 30 Table 2-4: Results of the SIMPER analysis showing the higher taxa contributing most to the similarity of the single group of 11 experimental rocks. Groups were ranked according to their average similarities (Av. sim.) and taxa according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given...... 34 Table 3-1: Sites on Mound 12 and related information of experimental wood blocks deployed on February 2009 and recovered after ~317 days by the manned submersible Alvin. Overall values of environmental variables measured in Mound 12 were: Temperature 5.1 °C, Oxygen (Winkler) 0.9-1.6 L-1, pH 7.6-7.7 (Levin et al., 2015). Act.=Seepage activity, A=Active, D=Depth, I=Inactive, ST&N= Substrate type and number. Wood #3 was lost...... 53 Table 3-2: Total abundance (standardized density, individuals in 10 cm-2 in parentheses) of meio-epifauna colonizing wood blocks for 10.5 months on Mound 12 - Costa Rica. Total percent (%) contribution of each group is also included. A=Active, I=Inactive, MB=Mussel bed, NMB=Near mussel bed, RB=Rubble, TW=Tubeworms. TA=Total abundance...... 59 Table 3-3: Dissimilarity results calculated by SIMPER comparing between seepage activity. Taxa contributing most to the dissimilarity between groups resulting from the cluster analysis. Av- XX Meio-epifauna Costa Rica

=average, Abund=abundance in the cluster group, Diss=dissmilatiry, SD=standard deviation, Contr=contribution, Cum=cummulative, A=Active, I=Inactive. Nematodes are the most important contributors to dissimilarity, being more abundant in woods located in inactive sites...... 60 Table 4-1: Habitat, survey dates, and depths of substrates deployed at 2009. A manned submersible recovered experimental substrates after ~317 days of deployment at Mound 12, Costa Rica. Other common characteristics to Mound 12 in this study: Temperature 5.1 °C, Oxygen (Winkler) 0.9-1.6 L-1, pH 7.6-7.7 (Levin et al., 2015). Act.=Seepage activity, A=Active, I=Inactive...... 80 Table 4-2: Abundance by copepod family colonizing each substrate after 10.5 months on Mound 12 - Costa Rica. Values are total number of individuals and density of individuals in 10 cm-2 (in parentheses) Total abundance (TA), and total percentage contribution (% C) of each family are also included (two last column). *A total of 24,279 individuals of Copepoda were obtained, but Clams#2 sample was not added to the total number of individuals presented in the table, as it was not possible to analyze it at the family level. A=Active, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, RB=Rubble, Sed=Sediments, TW=Tubeworms. Type and number simple of substrate: C#=Clams, R#=Rocks, T#=Tubes, W#=Wood...... 85 Table 4-3: Results of the various ANOSIM analyses comparing composition and density of meio-epifaunal copepod families colonizing for 10.5 months experimental substrates (Clams and tube substrates were not included in these analyses, only rocks and woods) located in six seepage active and inactive habitats and according to the background where they were located (inside and outside carbonate grounds). A=Active, BM=Bacterial mats, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, Sed=Sediments, TW=Tubeworms. *=significant...... 88 Table 4-4: Results of the SIMPER analyses showing copepod families contributing most to the similarity in activity (A=Active, I=Inactive), and habitats (Group I-Sed and Group A-NMB less than 2 samples in group). Groups were ranked according to their average similarities (Av. sim.) and families according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given...... 89 Table 4-5: Results of the Two-Way SIMPER analysis for similarity (using seepage activity and bed/carbonates as factors) and families that contributed to similarity. Stations (=activity-habitat combinations) grouped by seepage activity (A=Active, I=Inactive). Groups were ranked according to their average similarities (Av. sim.) and families according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), and cumulative percentage to similarity (Cum. %) are indicated. C=Carbonate background, NC=No Carbonate background. .... 96 Table 5-1: Locations (Mounds 11 ans 12), habitat, survey dates, and depths of substrates collection (natural rocks) in 2009 during AT 15-44 cruise by the Alvin manned submersible at Costa Rica. Act.=Seepage activity, A=Active, I=Inactive...... 110 Table 5-2: Taxonomic list and density (ind. 10 cm-2) by nematode genus at the different substrates studied of Mounds 11 and 12. A=Active, Bmat=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms. CG=% Contribution by genera, CF=% Contribution by family...... 112 Table 5-3: Univariate community indixes for Costa Rica Mound 11 and 12 nematode assemblages. S=total number of genera, N=Total number of individuals, d=Margalef’s species richness, J’=Pielou’s evenness, H’=Shannon Wiener’s diversity...... 115 Contents XXI

Table 5-4: Similarity results calculated by SIMPER (19 substrates). Taxa contributing most to the similarity between groups resulting from the cluster analysis. Also shown is the dissimilarity between the two main groups. Groups were ranked according to their average similarities (Av. sim.) and taxa according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given. Daptonema are the most important contributors to similarity in all groups. Groups A and B less than two samples in group. A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms...... 118 Table 5-5: Results of the varios ANOSIM analyses comparing composition and density of meio- epifaunal Nematoda between location, seepage activity, and habitat, for natural carbonate rock substrates. A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms. *= significant...... 120 Table 5-6: Contribution in abundance (%) of nematode feeding groups and Index of Throphic Diversity (ITD) by habitat (across localities). A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms.1A: selective deposit feeders, 1B=non-selective deposit-feeders, 2A=epistrate feeders, 2B=predators and/or omnivores...... 122

Introduction

The deep sea is recognized as the largest biome on Earth containing one of the highest levels of biodiversity as well as valuable biological and mineral resources. Nowadays, there is enough evidence documenting the presence of at least 28 different deep-sea habitats and ecosystems (Ramirez-Llodra et al., 2010). Of the above, environments based on chemosynthetic processes are recognized among the most important, because they represent a series of biological, chemical, and geological settings that host large biomass of highly specialized fauna with highly dynamic production processes.

Since the first seep communities were described in 1979 from San Clemente in California (USA), multiple discoveries of reducing environments in the continental margin have astonished deep-sea scientists. In Costa Rica, these communities were evidenced by Kahn et al. (1996) through observations of authigenic carbonates, bacterial mats, vestimentiferan tubeworms, mytilid mussels, solemyid and vesicomyid clams, in active and relict fluid venting sites along this accretionary site. After that, Levin et al. (2012) reported a composite hydrothermal seep ecosystem dominated by the siboglinid polychaetes Lamellibrachia barhami and Escarpia spicata, that represents an intermediate between the hydrothermal vent and cold seep ecosystems. This evidence suggests that the Costa Rican deep sea comprises a complex combination of reducing system attributes within the eastern Pacific margin (Levin et al., 2012). Of this area in particular, but for most of these systems, some faunal communities and associated processes are still unknown, becoming of great interest to understand at different scales their biodiversity, ecological trends, and the main factors that can influence their structure.

These reducing ecosystems are considered extreme environments. CAREX (2011) defines that “a natural environment is considered extreme when one or more environmental variables show values permanently close to the lower or upper limits for life”. Among other features, cold seeps are characterized by the presence of reduced chemical compounds (hydrogen sulfide, methane, and hydrocarbons), local hypoxia or anoxia, high microbial abundance and metabolic activity, and the production of autochthonous, organic material, by chemoautotrophic bacteria (Zeppilli et al., 2018). Cold seeps support a specialized community, structured by chemosymbiotic-bearing species that influence microscale diversity by supplying substrate, food, refuge, and a place for varied biotic interactions (Levin, 2005).

Continental margin heterogeneity exists in many forms and on multiple space and time scales and acts in a hierarchical, scale-dependent way to influence margin diversity at least in four levels (Menot et al., 2010). In this way, from a multidisciplinary point of view (i.e., oceanography, geology, 2 Introduction biology, microbiology, etc.), to understand at those different scales how these processes are involved and how they are correlated with margin diversity, becomes a primary objective. The four levels are:

▪ Largest scale: Hydrography associated with water masses and overlying productivity. ▪ Meso-scales (tens of kilometers): Topographic control in the form of canyons, banks, ridges, pinnacles, and sediment fans. ▪ Smaller scales: Earth and tectonic processes that control fluid seepage and sediment disturbance forming seeps. ▪ Smallest scales: Habitats formed by ecosystem engineers that influence diversity through the provision of the substrate, food, refuge, and various biotic interactions. They are represented by coral and sponge reefs, and mytilid, vesicomyid, and siboglinid beds. Also, the biotic influence can arise from decay processes from the whale, wood, and kelp falls.

In this sense, reducing environments contain a variety of features that are sources of biological and geomorphic heterogeneity. They can build large physical structures above the sediment surface to create habitat as foundation species for associated macro and meiofaunal communities (Bright et al., 2010). These foundation species are defined as large or spatially dominant organisms that create or provide habitats, colonized by other species. Foundation species influence the abundance, composition, and structure of the associated community and can provide food resources, living space, favorable settlement conditions, a refuge from predators, and/or from environmental stress (Bright et al., 2010). Both foundations species and their associated communities have been classified according to size categories (Thistle, 2003).

Meiofauna comprises the size class defined as the portion of the community passing through a 1 mm sieve and being retained on a 32 µm sieve (Bright et al., 2010; Vanreusel et al., 2010). This group of organisms has been the least documented even though they are very highly represented in the deep sea. Meiofauna can be found both as mud-dwelling infauna, and also as living epifaunally (or meio- epifauna) on surfaces ranging from large biogenic debris to seagrasses, macro-algae, larger coral fragments, sponge skeletons, manganese nodules, pebbles, etc. (Raes & Vanreusel, 2005). Meiofaunal communities are one of the most significant components in diversity and biomass in all microhabitats, and they have a critical role in linking bacterial and macrofaunal food webs (Gollner, 2009). This role could be highly significant in reducing environments because bacteria are the basis of the food-web as free-living organisms or as a symbiont of megafauna (Vrijenhoek, 2010).

Based on several studies, it has been established that meio-epifaunal communities on hard substrates tend to differ radically from those in adjacent sediments, and this would probably be because the conditions within the sediment are different from those on a complex elevated structure on the seafloor, regarding food supply, food quality, and the physical influence of strong bottom currents (Raes & Vanreusel, 2005). Therefore, these differences should have their effect on the presence and abundance of meio-epifaunal taxa. Also, small differences in microhabitat structure (i.e., smallest scales) should also influence meio-epifaunal community composition, as this group is known to respond acutely to minor environmental changes. In fouling processes, the dynamics of colonization and succession of these organisms and their role, remain almost unknown (Fonsêca-Genevois et al., 2006), especially in the deep sea, due to factors such as the high economic costs involved in observations over time, the technologies to be used, and in many cases, the lack of local expertise. Introduction 3

Although there is some knowledge related to metazoan meio-epifauna communities and colonization processes on biogenic structures, it is almost unclear how these processes occur on hard substrates, such as authigenic carbonates and sunken wood, and what are the ecological interactions involved. This work seeks to contribute to the knowledge of the deep-sea metazoan meio-epifunal community structure of seep areas, using as location the Costa Rican continental margin, an unexplored area of the Pacific, and approaching the problem from different levels. The analyses are mainly based on understanding (a) on a large scale (six locations) meio-epifauna colonization processes associated with inorganic substrates (authigenic carbonates - Chapter 2); (b) on a local scale (Mound 12 - wood blocks - Chapter 3), and (c) the influence of substrate on colonization processes and diversity, through focus on the two main representatives groups of meio-epifauna inhabiting on natural and experimental substrates, i.e., nematodes and copepods (Chapters 4 and 5).

A series of questions were explored according to the different worked scales.

▪ Are there linkages among meio-epifauna assemblages that inhabit different locations or habitats found at Costa Rica Cold seeps? ▪ Are the abundance, composition, and structure of the community associated to Costa Rica cold seeps influenced by foundation species? ▪ Does the assemblage structure of this community vary over time? ▪ Which factors (e.g., environmental, geological, hydrographic) could be affecting the micro- spatial structure of this fauna? ▪ Is the meio-epifaunal community in the Pacific of Costa Rica structured differently depending on the habitat?

1. Geological and biological background

1.1 Geological background

Since the beginning of research in the deep sea in 1844, when the azoic theory was proposed, it was thought that the deep sea was lifeless. However, it is recognized nowadays as the most significant biome on Earth, containing one of the highest levels of biodiversity as well as important biological and mineral resources. After nearly 175 years of hundreds of studies and research on the deep sea, there is much evidence documenting the presence of at least 28 different habitats and ecosystems (Ramirez-Llodra et al., 2010). These include deep-sea corals, canyons, hydrothermal vents, cold seeps, whale carcasses, etc. Within those mentioned above, environments based on chemosynthetic processes are recognized as among the most prominent ones because they represent a series of biological, chemical and geological settings that result in unique energy production processes. They are highly dynamic and support a large biomass of highly specialized fauna.

These reducing environments include hydrothermal vents, cold seeps, whale carcasses, oxygen minimum zones (OMZ), sunken wood, seagrass and other organic remains (Tarasov et al., 2005; Kaim et al., 2009). They have in common a chemosynthetic-based primary production (Van Dover et al., 2002). To cope with the anoxic conditions and to take advantages of the reducing power, they are inhabited by a wide variety of species exhibiting symbiotic associations with sulfur-oxidizing and methane-oxidizing bacteria (Duperron et al., 2005; Treude et al., 2011).

Deep-sea hydrothermal vents and cold seeps represent the two major ecosystems of this kind. These environments support a much higher standing crops biomass (in total biomass) than the surrounding deep sea, which makes them comparable to some of the most productive marine systems (Thurber et al., 2014). They significantly exceed values for the surrounding deep sea, where benthic biomass is of the order of a few grams per m2 (Sarrazin & Juniper, 1999; Bergquist et al., 2003). At cold seeps, most of the biomass is present as bivalves, and range between 2 and 20 kg m−2 (Tunnicliffe et al., 2003).

These reducing environments contain a variety of features that are sources of biological and geomorphic heterogeneity. For instance, vestimentiferans, frenulates, bathymodiolins, vesicomyids, other bivalves, sponges, microbial mats, oil seeps, mud volcanoes, salt diapirs, brine pools, gas hydrates, pockmarks, carbonates, and organic debris (Bright et al., 2010; Cordes et al., 2010). The shared requirement of hydrothermal vent and cold-seep communities is the presence of a reduced compound that can be oxidized by microbes to release energy for the fixation of organic carbon from

CO2 (or methane) (Van Dover et al., 2002).

6 Meio-epifauna Costa Rica

Particularly, cold seeps are known since 1979, when they were discovered on the active margin of San Clemente in California (USA) (Sibuet & Olu, 1998; Zeppilli et al., 2018). They are currently recorded in most of the margins of the world. In the eastern Pacific, they have been found at the Oregon Margin (Suess et al., 1985), north of Monterey Bay (Embley et al., 1990), Nicaragua (Buerk et al., 2010), Costa Rica (Kahn et al., 1996), Peru (Olu et al., 1996), and Chile (Sellanes et al., 2004). They are present in a wide bathymetric range from shallow waters (<15 m) off Santa Barbara, California (Levin, 2005) to hadal depths (9,345 m) in the Kurile Trench (northwest the Pacific Ocean) (Cordes et al., 2007).

The Pacific Ocean margins are typified by narrow continental shelves and steep slopes, frequently dissected by submarine canyons (Smith & Demopoulos, 2003). Present-day seafloor spreading in the Pacific basin involves nine oceanic plates: Pacific, Antarctic, Nazca, Cocos, and Juan de Fuca; also the Microplates Rivera, Galapagos, Easter and Juan Fernandez, along with the East Pacific Rise (EPR) (Bird, 2003; Seton et al., 2012). Primarily due to plate creation at the EPR, a large part of the Pacific Ocean margin consists of a series of subduction zones where the Pacific, Cocos and Nazca plates plunge into the Earth’s asthenosphere (i.e., weak ductile layer in the upper mantle, upon which lithospheric plates move) (Neall & Trewick, 2008).

To Costar Rica, a Country with coasts over the Caribbean Sea and the Pacific Ocean, its oceans represent an area of interest by its extension, geology, biogeography, and biodiversity richness, not only for the country but for the whole region. Its extension is reflected in the Pacific coastline with 1,254 km, harboring a great diversity of coastal and marine ecosystems, about six times the length of the Caribbean coast. Pacific waters of Costa Rica cover an area of 565,683 km2, representing 96 % of the marine area of the country (Cortés, 2016).

The Pacific continental margin offshore Central America shows a large number of cold seeps associated with faults, slump scars of submarine landslides, and mounds (Buerk et al., 2010). Sahling et al. (2008) revealed the presence of 112 seeps along the 580 km-long continental margin from southern Nicaragua to southern Costa Rica. For instance, the Costa Rican Margin is characterized by the presence of carbonates, mud volcanoes and gas hydrates (Cordes et al., 2010). Due to subduction process at the Costa Rica margin, the expulsion of methane-rich fluids and gases occurs over extended areas ranging in depth from 730 to 3,800 m (Levin et al., 2015). The Costa Rica margin hosts strong vertical hydrographic gradients. Between 400 m and 1,800 m, the temperature -1 ranges from 9.5 to 2.7 °C, bottom-water O2 concentration varies from 0.04 to 1.6 mL. L , and pH ranges from 7.7 to 7.8 (Levin et al., 2015). Bacterial mats are the dominant communities (Mau et al., 2006). For this research, substrates (natural authigenic rocks) were sampled in Mound 11, Mound 12, Mound Quepos, Jaco summit, Jaco scar, and Quepos landslide locations. Mound 12 was selected for a 10.5 months colonization experiment.

1.2 Meiofauna

The meiofauna is more diverse than any other component of the marine biota; 22-24 of the 35 metazoan phyla have representatives in the meiobenthos (Raes & Vanreusel, 2005; Balsamo et al., 2010), of which nematodes and copepods are the most representative in terms of both the number of species and abundance. In the deep-sea sediments, food supply is the factor with greater influence Chapter 1 7 in meiofaunal abundance and biomass, metabolic activity, community structure, reproductive cycles and size spectra (Raes & Vanreusel, 2005), while their horizontal distribution can be structured by the differentiated localization of trophic sources (Balsamo et al., 2010). Some other factors determining meiofaunal composition and diversity are the complexity of the biogenic structure, geochemical conditions, and bacterial processes (Van Gaever et al., 2009). In sum, it is the integration of physical, chemical, and biological variables, what shape abundance, composition, and distribution of meiobenthic and macrobenthic communities (Montagna et al., 2017).

Meiofaunal highest abundances are typically measured in intertidal muddy estuarine habitats, whereas the lowest are typically encountered in the deep sea (Balsamo et al., 2010). In shallow waters, abundance and biomass values vary according to the season, latitude, water depth, tidal exposure, grain size, habitat type, and other abiotic and biotic factors of the biotope (Balsamo et al., 2010). Meanwhile, towards the deep sea, all animal groups experience significant exponential decreases in both abundance and biomass. The density of deep-sea meiofauna inhabiting sediments is determined by three (sediment) factors: calcium carbonate content, heterogeneity of the substrate (low sorting coefficient), and organic matter, indicating food availability. Moreover, it is known there is a strong relationship between the meiofaunal abundance and biomass, and the supply of organic matter, which in turn depends on depth (Giere, 2009). However, it is still unclear which factors determine its composition and density of cold seep meio-epifauna.

In addition to being essential in the trophic web, meiofaunal organisms are ideal indicators for ecosystem health assessments (Montagna et al., 2017). A common concern when performing environmental assessments is the lack of baseline data previous to the events being studied (Montagna et al., 2017). Knowledge of meio-epifauna becomes essential, especially when deep areas are being subject to mineral and hydrocarbon exploration, which will intensify in the coming years.

Cold seeps can be considered as nutrient-rich islands with unevenly distributed sub-habitats, each harboring its specific epi and infauna (Van Gever et al., 2009). However, the knowledge of the biological assemblages that inhabit these environments and the potential ecological, biogeographic and phylogenetic linkages among those in different locations or habitats are limited (Metaxas & Kelly, 2010).

Several studies in extreme deep-sea environments have focused on megafauna, macrofauna, microbiology and associated geological processes. There are few investigations of metazoan meiofauna at cold seeps covering a variety of environments, water depths and geographic regions. In a broad sense, unconsolidated sediments have received most of the attention in macro and meiofaunal studies (Armenteros et al., 2012). In the eastern Pacific, meiobenthic and epifaunal community studies in deep waters are focused on the northern area. No studies on associated metazoan meio-epifauna off Costa Rica have been carried out so far.

1.3 Ecological relevance of colonization

The colonization hypothesis assumes that populations or organisms colonize new habitats because it is ecologically beneficial for them to disperse (Howe & Smallwood, 1982; Schranz, 2012). Schranz (2012) postulates several advantages of colonization, as follow: 8 Meio-epifauna Costa Rica

▪ The avoidance of inbreeding.

▪ The chance of locating a new site with low density of individuals and therefore few resource competitors.

▪ The possibility of escaping from unfavorable conditions.

▪ The genetic exchange between subpopulations within a metapopulation and enables dispersal of a species into new habitats or recolonization if a subpopulation is wiped out by disturbance.

In the deep sea, the interest in the processes of faunal colonization and succession can respond to two types of approaches: basic and applied (Smith & Hessler, 1987). Basic aims to identify the processes structuring, and the mechanism controlling, the initiation and maintenance of populations. Applied attempts to know, for example, how quick the ecosystem recovers after a mining process (Smith & Hessler, 1987), which is currently one of the main threats facing deep sea due to mining and hydrocarbon extraction. As these authors emphasize “Effective environmental management will require knowledge”. At present, many research efforts are being conducted worldwide to understand how these colonization processes have occurred and remain occurring in extreme deep-water environments.

1.4 Habitats and substrates

The biological communities that inhabit chemosynthetic environments face several challenges that arise from the peculiarities of the habitat (Metaxas & Kelly, 2010). “Meiobenthic community structure is regulated on small spatial scales (mm to cm) where patch dynamics are a function of biogenic structures, and conversely on larger scales (m to km) where benthic currents and shifts in sediment grain size regulate community structure” (Baguley et al., 2006). Meiofauna live on much smaller spatial and temporal scales, and mechanisms maintaining meiofauna diversity may be different than those for the larger-sized fauna (Baguley et al., 2006).

In deep sea, hard substrates are represented by authigenic carbonates, wood remains (that occasionally reach these depths and whose presence was documented years ago). Also, some animals, such as thyrasid bivalves, siboglinid vestimentiferans, bathymodiolin and vesicomyid bivalves, corals, and sponges can build large physical structures above the sediment surface. These hard substrates create habitat as foundation species for an associated macro- and meio-epifaunal community. In general, foundation species increase the surface area available and influence the abundance, diversity, species composition, and structure of the associated community (Plum et al., 2015); and can provide food resources, living space, favorable settlement conditions, and a refuge from predators and environmental stress (Bright et al., 2010).

At cold seeps, a variety of geologically diverse, reducing microhabitats, can be distinguished by the presence of microbial mats or macro/megafaunal communities (Bright et al., 2010). It is also possible to observe other types of microhabitats, provided by sunken woods or plant material that reaches these depths. Cold seeps are typically characterized by the presence of large megafauna aggregations, such as bathymodiolid mussels and siboglinid tubeworms. Chapter 1 9

Although cold seeps are now known to occur on continental margins throughout the global ocean, variations in the composition of seep communities in different parts of the world are not well resolved (Sibuet & Olu, 1998; Bowden et al., 2013). Only a small fraction of the bottom oceans have been studied and explored worldwide. Therefore, these studies contribute to a better understanding of the various processes associated with Cold Seeps in the Pacific Ocean.

1.5 General methodology

1.5.1 Field methods

Primary information of this study was obtained during two research expeditions performed by the Scripps Institution of Oceanography - UC San Diego, on board R/V Atlantis and using the manned submersible Alvin. From the first expedition, cruise AT 15-44, substrate samples (natural rocks) were collected from different associated habitats of cold seeps off Costa Rica, and colonization experiments were deployed (see below). During the second expedition, cruise AT 15-59, all colonization experiments were retrieved from the field. Chapters 2 to 5 describe methodologically how the information from the meio-epifauna was analyzed.

Habitat sampling. Within the framework of the research expedition AT 15-44 carried out in 2009, 41 carbonate rocks suitable for meio-epifaunal analysis were collected between 376 to 1800 m in depth from six different locations, i.e., Mound 12, Mound 11, Mound Quepos, Jaco wall, Jaco summit and Quepos landslide. Habitats included mussel beds, tubeworm bushes, bacterial mats, clam beds and sediments.

Colonization experiments on Mound 12. Twenty-five pieces of four different substrates were deployed on February 22-23 and March 5, 2009 on active and inactive areas on Mound 12. This experiment consisted of placing bare substrates such as authigenic carbonates, wood, and biogenic material on the sea-floor. These substrate types were selected as representative of hard substrates at a reducing ecosystem on the Costa Rican margin, including methane seeps, sunken wood falls, and seep-specific tubeworms, mussels, or clams. The depth of the experiments was about 1000 m on the Costa Rica margin.

Recovery of colonization experiments and sample preservation. Experimental substrates were recovered by manned submersible after about 317 days of deployment, January 7-10, 2010. Substrates were retrieved by the mechanical hand of the submersible and placed into an insulated biobox with Plexiglas compartments that maintained separation of fauna. On board, substrates were thoroughly washed with filtered sea-eater and the water passed through 300 and 43 µm sieves to recover meio-epifauna. The meio-epifauna fraction was fixed in 8 % buffered formalin.

Environmental variables. Hydrography was examined with CTD casts and sampled bottom water. Variables measured were oxygen, temperature, pH, and depth.

2. Meio-epifaunal colonization on carbonate substrates in deep cold seeps of the Costa Rican Pacific margin

Abstract

Carbonate authigenic substrates, naturally present in deep-sea habitats, play an important ecological role in cold seep environments. They provide hard ground for settlement, enhance complexity of the habitat, and supply shelter and food, among other roles. An ecological survey was carried out to study higher taxonomic rank taxa assemblages of meio-epifauna found on 28 natural carbonate rocks collected at the Costa Rican Pacific continental margin associated with methane seeps. Spatial community variation in relation to methane seepage activity and habitats, and hydrographic conditions (depth, temperature, oxygen, pH) were studied. Six different locations (Mound 12, Mound 11, Mound Quepos, Jaco wall, Jaco summit, and Quepos landslide), located between 344 and 1856 m in depth, were sampled using the manned submersible Alvin during R/V Atlantis cruise AT15-44 - 2009. In each locality, pre-defined habitats within seep areas (seepage active) and next to them (seepage inactive) were sampled for loose rocks (from large cobble to small boulder size). Six activity-habitat combinations were sampled, i.e., inactive: carbonate rocks-hard corals, and rocks found on sediments; and active: tubeworm bushes, bacterial mats, near clam beds, and mussel beds. Additionally, pre-processed carbonate rocks were deployed at Mound 12 (~997 m in depth) for a 10.5-month temporal colonization experiment, and then recovered during R/V Atlantis cruise AT15- 59 - 2010. A total of 27 meio-epifaunal taxa formed the assemblage of natural rocks, many more than common deep-sea biogenic substrates. Nematodes (58.1 %) followed by copepods (24.9 %) were the most abundant. Inactive areas exhibited the highest mean number of individuals (19.2±17.6 (1 SD) ind.10 cm-2, n= 17), while in active sites the mean was 13.1 ± 13.7 ind.10 cm-2, n = 11. Significant differences in higher taxa composition and abundance were found between locations (Jaco scar, Jaco wall, Mound 11, Mound 12, Mound Quepos, Quepos landslide) and between habitats (Bacterial mats, carbonates, hard Coral, mussel beds, near clam beds, Oxygen Minimum Zone, sediments, tubeworms), but not between seepage activities. Different locations exhibit different hydrographic characteristics, with depth and temperature being the variables that best explain the biological scheme found. After10.5 months of colonization of authigenic rocks, the community was comprised of 14 meio-epifaunal taxa. In contrast to natural rocks, copepods accounted for the highest abundances (69 %) on colonization rocks, followed by nematodes (18 %). As in natural rocks, the highest mean density was found in inactive areas (26.3±11.3 ind. 10cm-2, n=5) compared to active areas (16.4±16.4 ind. 10cm-2, n=6). Also, significant differences were found between habitats but not between seepage activities. Contrary to previous studies of deep-sea hard bottom meio-epifauna,

12 Meio-epifauna Costa Rica in which crustaceans have been found to be the dominant component, the natural carbonate rocks studied here were dominated by nematodes. Their known special adaptations for dwelling on hard surfaces need to be considered in detail. In addition, the dominance of copepods during the initial months of colonization warrants a closer look.

Keywords: Authigenic carbonate rocks, Substrates, Colonization, Extreme environments

2.1 Introduction

A major component of life in the deep sea relies on chemosynthetic energy from fluids emitted at hydrothermal vents and cold methane seeps, where prokaryotes consume sulfide or methane for primary production, supporting oasis-like ecosystems rich in biomass of animals (Levin, 2005; Cordes et al., 2010, Sapir et al., 2014). Within the fauna associated with these ecosystems, various components are classified according to their size and associated function, i.e. megafauna, macrofauna, meiofauna and microfauna. Species and colonization process about mega and macrofauna are better understood, while the roles of the smaller size components are still largely unknown.

One of the concerns that arises when trying to understand the diversity of organisms associated with deep-sea extreme environments is how their communities develop and what are the main factors that intervene in their colonization process, whether on hard surfaces or within the sediments. In deep water bottoms, a diversity of substrates are found, both of organic and inorganic origin, i.e., sunken woods, animal bones, authigenic carbonates, coral skeletons, mollusks shells and polychaete tubes, among others. These are suitable for the development of small sized organisms, which in turn provide trophic support to larger sized animals, following a complex trophic pattern.

Colonization processes on surfaces begin as soon as they are submerged or exposed in the ocean, being immediately subjected to an initial fouling process (Rittschof, 2000). For this process of colonization, three levels of organization have been recognized: a) Molecular fouling, b) Microfouling, and c) Macrofouling. That is, colonization begins then with the accretion of organic and/or inorganic molecules from solution onto submerged surfaces. Subsequently, the colonization of surfaces by micro-organisms occurs, followed by the secretion of polymers that anchor and often embed the micro-organisms and other particles. And this sequence ends with the colonization of surfaces by macroscopic fauna and flora, usually by means of micro- or meiosized propagules (Rittschof, 2000; Fonsêca-Genevois et al., 2006). However, these sequences are not always linear; several studies have found that microfouling is not a prerequisite for macrofouling (Fonsêca- Genevois et al., 2006).

There are species that provide habitat for others, known as foundation species, which have the capacity to modify their physical and chemical surroundings. These include kelp forests, mussel beds, oyster reefs, coral reefs, etc. (Bruno & Bertness, 2001). By their very nature, they can build large physical structures above the sediment surface, and can provide food resources, create or provide habitats for associated macro and meiofaunal communities, provide refuge from predators, and/or refuge from environmental stress, and facilitate the arrival of other species by increasing propagules retention; they influence abundance, composition, and structure of the associated Chapter 2 13 community (Bruno & Bertness, 2001; Bright et al., 2010). These foundation species are defined as large or spatially dominant organisms; they tend to be sessile, and their interaction with other species is often defined by their presence, rather than their actions (Govenar, 2010). How large a foundational species needs to be to provide habitat, however, depends on the scale at which the heterogeneity is evident (Levin, 1992; Cordes et al., 2010). Reducing environments in the deep sea contain a variety of features that are sources of biological and geomorphologic heterogeneity, foundation species being one of them. In cold seeps, several species are known to operate as foundation species, among them large bivalves and polychaete tubeworms.

Meiofauna comprise the size class defined as the portion of the community passing through a 1 mm sieve and being retained on a 32 µm sieve (Bright et al., 2010; Vanreusel et al., 2010). These meiofauna are characterized by their small size, short life span and thus high species turnover, and lack of larval dispersal in their life cycles. This group of organisms has been the least documented and understood even though they are well represented in the deep sea. Meiofauna can be found both as mud-dwelling fauna, and also as group living epifaunally (or meio-epifauna) on surfaces ranging from large biogenic debris such as seagrass blades, macro-algae, larger coral fragments, sponge skeletons, tubeworms, echinoderm spines, manganese nodules, pebbles, sea ice, etc. (Coull, 1988; Raes & Vanreusel, 2005). In addition to the importance of knowing the role of meiofauna in the trophic web, the knowledge of which organisms inhabit this kind of environment and their ecological interactions is the first step towards its management and conservation. These organisms can be ideal indicators for ecosystem health assessments (Fraschetti et al., 2006; Montagna et al., 2017). A common concern when performing environmental assessments is the lack of baseline data previous to the events being studied (Montagna et al., 2017). As already mentioned, very few attempts have been made to investigate meiofaunal assemblages associated with deep-sea environments.

Meiofauna are generally described as featuring heterogeneous spatial patterns of distribution (Coull, 1988; Steyaert et al., 2003), but the quantification of this patchiness is rarely conducted by using appropriate experimental designs (Fraschetti et al., 2006). Meiofauna are dependent upon the characteristics of the substrate (Donavaro & Franchetti, 2002). As mentioned by several authors (summarized in Fonsêca-Genevois et al., 2006) “there are known significant differences between meio-epifauna communities on hard substrates to those in neighboring sediments, and the dynamics of colonization and succession of these organisms and their role, in fouling processes, remain partly unknown”. The above becomes more relevant as cold seeps have been considered among the most heterogeneous habitats of continental margins (Cordes et al., 2010). This heterogeneity is provided by the very contrasting conditions in their hydrography (depth, temperature, oxygen), geochemical characteristics (gas activity, fluid flow intensity and volume), geological features (tectonic activity, mud activity, diverse geomorphology, authigenic carbonates, geologic features age), and biological composition (microbial communities, foundations species) (Cordes et al., 2010).

Understanding colonization processes in deep water meio-epifauna is challenging, mainly due to their small size and the associated effort in their collection and study; the deep-sea conditions are extreme, and the hard bottoms are small and patchy in the vastness of the deep ocean. Carrying out experiments of colonization and succession in the short and medium term, requires logistics that are often not available. However, more inter-institutional efforts are being made to resolve ecological questions that still remain about this group of organisms. In deep-sea methane cold seeps, the focus 14 Meio-epifauna Costa Rica has been on those organisms associated with the different constituent habitats, such as sediments, loosely consolidated carbonate protoliths, fully lithified carbonate blocks and pavements, and, occasionally, the woody debris that reach those depths (Case et al., 2015). All these habitats offer a range of surfaces that harbor a diverse fauna, from microscopic microfauna and meio-epifauna, to large sized macrofauna and megafauna. The knowledge of the ecology of these organisms in many cases requires understanding the colonization processes and the relationships with environmental factors that limit their occurrence.

A conspicuous characteristic of Costa Rican Pacific cold seeps is the presence of authigenic carbonates. Their precipitation is driven by the anaerobic oxidation of methane (AOM) which is mediated by consortia of methane-oxidizing Archaea and sulfate-reducing bacteria; they are formed as a result of increased alkalinity associated with AOM metabolism (Han et al., 2004; Pierre & Fouquete, 2007; Case et al., 2015). These carbonate substrates occur in a variety of sizes, morphologies, and mineralogies (Case et al., 2015). Several types of authigenic carbonates have been recognized throughout various areas of the Costa Rican margin, such as chemoherm carbonates (Mound 12, Jaco scarp), seepage-associated concretions (Mounds 12 and Quepos, Quespos slide), gas hydrate-associated concretions (Mound 11), and calcareous and dolomitic concretions (Mounds 11, 12, Quepos, Jaco scarp), relying on their morphology, mineralogy, petrology and C and O isotopic compositions (Han et al., 2004). The main characteristics and functions of these carbonates are: (1) they have very low d13C values (down to –62‰ V-PDB Vienna Pee Dee Belemnite standard), owing to the methane source of carbon (Pierre & Fouquete, 2007); (2) they host lipid and ribosomal DNA biomarkers as evidence for anaerobic oxidation of methane; (3) they host viable autoendolithic (organisms whose metabolism induces self-entombing mineral formation) Archaea and Bacteria capable of methane oxidation; and (4) host metazoan communities (Stadnitskaia et al., 2005; Case et al., 2015). Authigenic carbonate environments harbor methane-dependent seep fauna, along with various other types, depending on the substrate and the local distribution of fluids (e.g., Feng & Chen, 2015; Liang et al., 2017). These faunas may function as environmental indicators, because physical, chemical, and biological processes at seeps can change significantly with time (Feng & Chen, 2015).

Meiofaunal colonization studies in cold seeps have been mainly conducted on biogenic substrates (Bright et al., 2010; Degen et al., 2012). Studies on inorganic substrates, like carbonates or rocks, are relatively scarce, with emphasis on other deep-sea ecosystems, such as cold water coral bottoms (Raes & Vanreusel, 2005) or hydrothermal vents, either using panel arrays (Van Dover et al., 1988), artificial tubeworm aggregations (i.e., low-grade polyvinyl chloride hose) (Govenar & Fisher, 2007), experimental carbonate cubes (Gaudron et al., 2010, also on cold seeps), sponge-like complex structures with interstitial spaces, and a basalt mimic (Kelly & Metaxas, 2008), or basalt-like slate substrates (Zeppilli et al., 2015), among others. For our study area, the information on colonization process comes from recent efforts, and what is known includes larger organisms (macrofauna). Levin et al. (2015) found that, at a coarse taxonomic level, the composition of carbonate macrofauna bears remarkable resemblance to the biota of temperate and tropical rocky intertidal shorelines. Bacteria attached to carbonates appear to support high densities of grazing coiled snails and limpets, just as microalgae (and Cyanobacteria) do on rocky shores. However, this resemblance has not been established for meio-epifauna. Chapter 2 15

In the eastern Pacific, the Costa Rican continental margin has heterogeneous areas with different combinations of reducing systems. Like in other extreme deep-sea environments, these reducing communities are highly specialized, structured by chemosymbiont-bearing species that could influence microscale diversity throughout supply of substrate, food, refuge, and various biotic interactions (Menot et al., 2010). In the same way, carbonate precipitation is a widely observed phenomenon in this seep environment (Cordes et al., 2010; Levin et. al., 2015). All of the above (heterogeneity, foundation species, carbonates) provide a scenario to understand, at different geographic scales, the colonization processes of the meio-epifauna. In this work, we undertook manned submersible collections of authigenic carbonate rocks from six different locations in active and inactive sites off the Costa Rican Pacific continental margin, to investigate the community structure of their metazoan meio-epifauna. The main aim was to answer the question: How do seepage activity-habitat type combinations (inactive: carbonate rocks, rocks on sediments; active: tubeworm bushes, bacterial mats, near clam beds, mussel beds) and hydrographic conditions (depth, temperature, oxygen, pH) influence meio-epifuanal assemblages associated with carbonate rocks? In addition, in order to begin with the understanding of the early colonization phase of meio- epifauna, clean carbonate substrates were deployed in one location and retrieved 10.5 months later for study. This is the first study to evaluate the composition of meio-epifauna on inorganic substrates in Costa Rican deep cold seeps.

2.2 Material and Methods

2.2.1 Study area

The Pacific continental margin offshore Central America shows a significant number of cold seeps associated with faults, slump scars of submarine landslides, and mounds (Buerk et al., 2010). For instance, the Costa Rican margin is characterized by the presence of carbonates, mud volcanoes and gas hydrates (Cordes et al., 2010). These carbonates, found between 800 and 1500 m in depth, reflect both thermogenic and biogenic methane sources (Han et al., 2004; Levin et al., 2015). The study area in general is colonized by chemosynthetic communities including Calyptogena clams, Lamellibrachia tubeworms and Beggiatoa bacterial mats (Burkett, 2011), these bacterial mats being the dominant communities (Mau et al., 2006).

For our study, material was collected from six different locations, i.e., Jaco summit, Jaco wall, Mound 11, Mound 12, Mound Quepos, and Quepos landslide (Fig. 2-1). Some characteristics of each sampling location are described below. Special emphasis was placed on Mound 12, where both natural and experimental rocks were used. According to Levin et al. (2015), locations are within the -1 oxygen minimum zone – OMZ where there is < 0.5 mL.L O2 (Quepos landslide; ~380 m), at the lower boundary of the OMZ (Jaco summit; ~740m), and below the OMZ (Mounds 11, 12, Quepos, and Jaco wall; ~970-1856 m).

Mound 11. This low relief mud volcano, about 20 m high (Mau, 2004), is located at 1017 m in depth, and characterized by carbonates, gas hydrates, bacterial mats, and siboglinid tubeworm bushes (Sahling et al., 2008). Gas hydrates and methane are primarily of thermogenic origin (Sahling et al., 16 Meio-epifauna Costa Rica

2008). Bacterial mats are the dominant community, occupying an area of 500-1700 m2 (Mau, 2004). Seepage fluid fluxes at Mound 11 are the highest measured at the continental margin of Costa Rica (Sahling et al., 2008).

A

B C D E

Figure 2-1: A. Study locations on cold seeps on the Costa Rica continental margin in the Pacific Ocean. B-E. Photos of some substrate samples taken on board immediately after retrieval (with permission from Greg Rouse), B. Jaco wall, C. Mound 11, D. Mound 12, E. Mound Quepos. Cruise AT-15-44. For scale see the hand holding the rocks.

Mound 12. This is an active low relief mud volcano, 30 m high, located at 1020 m in depth, elongated in northeast-southwest direction with diameters of about 1 to 1.6 km (Mau, 2004, 2006; Niemann et al., 2013). This mound is characterized by the presence of high methane concentrations in the water column, authigenic carbonates, and chemosynthetic communities (Crutchley et al., 2014). Mound 12 seems to be most active at its pinnacle and the SW flank, which is characterized by dense microbial mats and other chemosynthetic organisms (mytilid mussels and Lamellibrachia tubeworms) (Niemann et al., 2013). According to Mau et al. (2006) at Mound 12 bacterial mats are the dominant community, occupying an area of 1500-5000 m2. At this mound benthic fluxes are high in sediments covered by bacterial mats (Sahling et al., 2008). Chapter 2 17

Mound Quepos. Located further north of Mounds 11 and 12 at ~1400 m in depth, it is characterized by extensive fields of authigenic carbonates, vesicomyid clams, mytilid mussels and vestimentiferan tubeworms (Lamellibrachia barhami) (Sahling et al., 2008).

Jaco wall. Located at 1850 m in depth, it is a ‘hydrothermal seep’, an intermediate between a cold seep and hot vent, with characteristics of both (Levin et al., 2012). Methane concentrations were high (190-860 nM) above the bottom, at 1764-1734 m in depth (Levin et al., 2012).

Jaco summit. Water collected at ~745 m had methane concentrations less than 15 nM (Levin et al., 2012). No extensive information in the literature on this geoform was found.

Quepos landslide. This geoform, starting at ~400 m in depth, is characterized by an oxygen- minimum layer in the water column between 200-600 m in depth (Bohrmann et al., 2002). In this location the chemosynthetic communities are extensive, along a 10-km wide area of the slope (Sahling et al., 2008). Bacterial mats were observed covering an area of tens of m2 on top of very soft sediments, immediately below the headwall at depths of around 400-410 m, which coincides with a methane concentration of 75 nmol L–1 around 402 m (Bohrmann et al., 2002; Sahling et al., 2008). Authigenic carbonates were observed exposed on the crest of a small ridge (Bohrmann et al., 2002).

4586 B

4587 C

#2

4588 D

A

Figure 2-2: A. Habitats where experimental rock substrates were deployed on Mound 12 for 10.5 months of colonization on Cruise AT-15-44, with recovery on AT 15-59. B-D. Photos of some experimental rocks after retrieval (numbers indicate diving logs; pictures with permission from Greg Rouse). A=Active, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, RB=Rubble, SED=Sediments, TW=Tubeworms. 18 Meio-epifauna Costa Rica

2.2.2 Habitat sampling

Primary information for this study was obtained during two research expeditions performed by the Scripps Institution of Oceanography - UC San Diego, on board R/V Atlantis and using the manned submersible Alvin. Sampling was posible through a collecting permit issued by the Costa Rica “Ministerio de Ambiente y Energía, Sistema Nacional de Áreas de Conservación” (Levin et al., 2015).

Collection of natural rocks. From the first research expedition, cruise AT 15-44 carried out in 2009, 28 loose carbonate rocks suitable for meio-epifauna study, ranging in size from 180 to 2,174 cm2 of surface area, were collected from different habitats associated with cold seeps (Appendix 1). Also, during this cruise, the rocks for the colonization experiment were deployed (Fig. 2-2). Natural rocks were collected between 344 to 1856 m depth from the six locations described above. Active sites within locations were defined by the presence of seepage, indicated by bubbling, bacterial mats, and chemosynthetic communities. Sites with no signs of fluid flow or chemosynthetic macrofauna were defined as inactive. For the present analysis, no rocks were obtained from both active and inactive sites for all locations; this was possible only in Mound 11, Mound 12 and Jaco wall. Number of rocks collected differed among active and inactive sites depending on their availability. Inactive habitats included carbonate grounds-hard corals (I-Car), and rocks on sediments (I-Sed), while active habitats were tubeworm bushes (A-TW), bacterial mats (A-BM), near clam beds (A-NCB), and mussel beds (A-MB); some habitats were located within the OMZ. There were 14 different combinations of location, habitat and seepage activity, and in each there were 1 to 5 substrate samples (Appendix 1, Fig. 2-3).

Colonization experiments on Mound 12. The carbonate rocks used in the 10.5-month experiment were obtained either at methane seeps of Hydrate Ridge, Oregon (44°40’N, 125°6’W) or collected from Mound 12. They were pre-processed (defaunated and dried in the open for one week) and then deployed (Grupe, 2014). Rocks were deployed in duplicate (except in soft sediments where only one rock was deployed). Eleven rocks (ranging in size from 562 to 1,372 cm2) were deployed on the sea- floor on 22-23 February and 5 March 2009 on Mound 12 (cruise AT 15-44), in six activity-habitat combinations (inactive: rubble field, sediments, near mussel beds; active: tubeworm bushes, mussel beds, near mussel beds) (Appendix 2). The same criteria for natural rocks were used to define active and inactive sites. The depth of the experiments was about 1000 m (Fig. 2-2). During the second expedition, from 7 to 10 January 2010, cruise AT 15-59, all colonization rocks were retrieved by manned submersible, having been exposed for 317 days (10.5 months). During retrieval, rocks were placed into an insulated biobox with Plexiglas compartments that maintained separation of fauna.

2.2.3 Density quantification

On board, sample substrates were washed with filtered sea water, and the liquid residual was sieved through 300 and 43 µm meshes to recover meio-epifauna. Organisms included meio-epifauna and some elements of meiofauna which by their size are part of the macrofauna (e.g., big sized nematodes). The meio-epifauna fraction (retained by a 43 µm mesh net) was fixed in 8 % buffered formalin. To separate specimens from other material, the filtered sample was Rose Bengal stained Chapter 2 19 and placed onto Ludox HS 40 (density 1.31 g cm-3) 5-6 times, for density decantation, following D. Leduc-Meioscool (2013).

Metazoan meio-epifauna was manually sorted and counted under a ZEISS Discovery.V8 stereo microscope, and identified to a higher taxonomic category (e.g., Nematoda, Copepoda, Polychaeta, Ostracoda, Kinorhyncha, Bivalvia, Gastropoda, nauplii, etc.) following Higgins & Thiel (1988) and other complementary literature. Unknown organisms were assigned to the group “others”. The presence of crustacean nauplii (overall, as it was impossible to assign them to a specific crustacean taxon) was included because they may provide ecological information about the composition of the community, and because this category appeared constantly and numerously in all samples. Some meio-epifaunal organisms were then preserved in 4 % buffered formalin (e.g., nematodes), and others such as mollusks and ostracods were preserved in 70 % ethanol.

2.2.4 Data analyses

Composition and abundance. To calculate carbonate rocks surface area, all rocks pieces were covered with a single layer of aluminum foil, which was later weighed, and surface area calculated given the mass of a 5 x 5 cm foil square. To all substrate samples (natural and experimental) the area ranged between 180 to 2,174 cm2. The density was calculated from abundance and substrate surface area. In order to compare among environments and with other meio-epifaunal studies, total abundance was standardized to number of individuals per 10 cm-2 of rock surface area. Data were organized as taxa x location, seepage activity, and habitat matrix.

Community patterns. To describe the structure of the meio-epifaunal assemblages, for both natural and experimental rocks (including locations, seepage activity, habitats), multivariate analyses were performed. A similarity matrix was produced using the Bray-Curtis similarity measure, from fourth root transformed data, to down-weight high abundance groups. The matrix was used for hierarchical agglomerative clustering using group average sorting, and for Multidimensional Scaling Ordination (MDS). Data from individual substrates were used to establish patterns. According to the clusters obtained, a Similarity Profile Analysis (SIMPROF, Type 1, Significance level 5 %) was carried out to determine significant cluster structure.

For the 28 natural rocks, to determine the possible significant difference in meio-epifaunal density due to locations, seepage activity, and habitat, a series of one-way analyses of similarity (ANOSIM with 9999 permutations, and a p<0.05 significance level) were carried out. For this, a Bray Curtis similarity matrix was created with fourth root transformed density data. As habitats were nested within activities for natural rocks, to test whether activity or habitat had an effect on the density of colonizing meio-epifauna, a two-way nested ANOSIM was made. These tested the following hypothesis: There were no differences in the composition and density of meio-epifauna between locations, there were no differences in the composition and density of meio-epifauna between seepage activity, there were no differences in the composition and density of meio-epifauna between habitats. The Similarity Percentage (SIMPER) test (cut-off of 50 %) was next performed in order to determine the contribution of each taxa to the total dissimilarity. 20 Meio-epifauna Costa Rica

According to the groups obtained, statistical comparisons of densities and number of species were made using the non-parametric test of Kruskal-Wallis (one-way analysis of variance by ranks data). This is used to determine statistically significant differences between two or more groups of independent variables. The test was performed using PAST3 (Hammer et al., 2001). This information was related to environmental variables in order to detect any patterns.

In order to relate the distribution of the meio-epifaunal assemblages and the environmental variables (depth, temperature, oxygen, pH) for natural rocks, a RELATE test (Spearman rank) and a BEST- BIOENV (Spearman correlation) procedure were used. The environmental variables were transformed (fourth root) and normalized. The RELATE test shows the significance of relationship between the biological variables (taxa abundances) and environmental variables matrix (this is a non-parametric form of the Mantel test). BEST-BIOENV calculates which variables are the best correlated between biological data and individual variables; the maximum coefficient obtained from all the possible combinations indicates that this is the one that 'best explains' the biological scheme obtained in assemblages. These analyses were performed using PRIMER (Plymouth Routines in Multivariate Ecological Research) v. 7.0 programme.

For experimental rocks, the same methodological design as explained above was followed. However, no comparison or analysis by locality was made, because the experiment was carried only at Mound 12. As for experimental rocks there was one habitat (near mussel beds - NMB) in which there were active and inactive sites, a 1-way ANOSIM comparing activities was carried out.

2.3 Results

2.3.1 Composition and abundance of natural rocks

Twenty-seven phyla/subdivisons, including permanent and temporary meio-epifauna were collected. Permanent meio-epifauna (i.e., Nematoda, Copepoda, Chaetognatha, Cladocera, Copepoda, Halacaroidea, Isopoda, Kinorhyncha, Nemertina, Oligochaeta, Ostracoda, Sipunculida, Tanaidacea, and others) accounted for approximately 90 % of the total number of organisms, while temporary meio-epifauna represented the remaining 10 % (Appendix 3). Nematoda, Copepoda, nauplii larvae, Polychaeta, Gastropoda, Ostracoda and Bivalvia were among the most representative groups. Figure 2-3 presents the information of the 28 natural rocks collected, averaged by habitat (n varies from 1 to 5). The dominance of nematodes in almost all habitats was clear.

A total of 22,956 individuals (Appendix 3) of meio-epifauna were found associated with carbonate rocks in the six different locations, between 344 and 1,856 m in depth. Total mean meio-epifaunal density (28 substrates) was 16.8±16.2 ind.10 cm-2 (mean±1 SD).

Figure 2-4 shows the contribution (% ind. 10 cm-2) of different groups to the total meio-epifauna found in natural rocks (A) and the number of taxonomic groups (B) regardless of activity or habitat. Quepos landslide (the only OMZ site of this study) is highlighted by the highest relative abundance of nematodes (and the very low abundance of copepods) (Fig. 2-4 A) compared to the other groups, although with the lowest mean number of taxa (i.e., 14 in Jaco Summit, 13 in Mound 11, 11 in Jaco wall and Mound Quepos, 10 in Mound 12, and 8 in Quepos landslide) (Fig. 2-4 B).

Inactive-Carbonates Inactive-Sediments Active-T. worms Active-B. mats Active-NCB Active-MB

n=2 n=2 n=2 n=1 n=1 n=5 n=1 n=3 n=1 n=1 n=1 n=3 n=1 n=4

100% Chaetognatha Caudofoveata 90% Tanaidacea Solenogastres Sipunculida 80% Pycnogonida Polyplacophora 70% others Ophiuroidea Oligochaeta 60% Nemertina Larvae 50% Larvacea Kinorhyncha Isopoda 40% Halacaroidea Cnidaria

Relative abundance (%) abundance Relative 30% Cladocera Brachiopoda 20% Amphipoda Bivalvia Ostracoda 10% Gastropoda Polychaeta 0% Nauplii Copepoda Nematoda

Figure 2-3: Relative abundance (% of total density) of different taxonomic groups of meio-epifauna found in natural rocks as function of habitat (marked at the top of the bars). A=Active, BM=Bacterial mat, BoulF=Boulder Field, Carb=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco summit, JW=Jaco wall, MB=Mussel beds, MQ=Mound Quepos, M11=Mound 11, M12=Mound 12, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, TW=Tubeworms. n=number of substrates per location.

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80% Pycnogonida Polyplacophora others 70% Ophiuroidea Oligochaeta Nemertina 60% Larvae Larvacea 50% Kinorhyncha Isopoda Halacaroidea 40% Cnidaria

Cladocera Relative abundance (%)abundance Relative Brachiopoda 30% Amphipoda Bivalvia 20% Ostracoda Gastropoda Polychaeta 10% Nauplii Copepoda

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Figure 2-4: A. Contribution (% ind. 10 cm-2) of different groups to the total meio-epifauna found in each location on natural rocks. B. Mean (±SD) number of taxa found in each location.

Chapter 2 23

Natural rocks also were examined for the presence of any tendency in the number of taxa and in the density according to the habitat, in order to determine if foundation species could structure the assemblage increasing diversity or density of organisms. Regarding the number of taxa, similar results were found on average, I-Sed had 10.8 (±3.2) taxa, I-Car 12 (±2.4) taxa, A-MB 9.5 (±2.1) taxa, A-NCB 14 taxa, A-TW 11.0 (±2.8) taxa and A-BM 10.3 (±1.7) taxa. Figure 2-5 shows no clear pattern in density associated with foundation species presence (i.e., TW, BM, MB). NCB showed the highest density and the highest number of taxa, but it only corresponds to one rock substrate, so no pattern can be established.

In grouping by seepage activity, the highest mean density was found in inactive areas (19.2 ± 17.6 ind.10 cm-2, n = 17), while in active areas it was 13.1 ± 13.7 ind.10 cm-2, n = 11. According to the habitat, I-Sed (located in OMZ) had the highest number of associated individuals, with 76.8 ind.10 cm-2 (replicas support that pattern) (Appendix 3).

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Figure 2-5: Contribution (% ind. 10 cm-2) of different groups to the total meio-epifauna found in each habitat on natural rocks. A=Active, BM=Bacterial mat, Carb=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, TW=Tubeworms.

2.3.2 Community patterns of natural rocks

Group cluster analysis and multi-dimensional scaling (MDS) ordination are shown in Figs. 2-5 A, B. The dendrogram, plus the SIMPROF test, closely correspond to the NMDS cluster. SIMPROF support five main groups (A to E), two of them (C and D) composed of a single substrate; one of the latter (D) is merged to group E in the MDS ordination plot. These analyses revealed that metazoan meio-epifauna discriminated at higher taxonomical ranks do not clearly cluster or segregate by one particular factor, location or activity-habitat. There are, however, some trends. Regarding locations, Quepos landslide (QLS) substrate samples (all coming from inactive sediments), which are 24 Meio-epifauna Costa Rica characterized by the high densities of organisms, especially nematodes (Fig. 2-3), and by being located in an OMZ and being the shallowest (344-380 m depth), clearly separate from the other substrates; also, all eight Mound 11 (M11) substrates are within the same SIMPROF homogeneous cluster (Fig. 2-6 A) and a 65 % similarity group in MDS (Fig. 2-6 B). In addition, there is a trend related to activity-habitat as all but one of the six non-QLS inactive-sediment associations are grouped together.

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Non-metric MDS Transform: Fourth root Resemblance: S17 Bray-Curtis similarity 2D Stress: 0,16 Similarity M12-I-Sed M12-A-TW 65 M12-A-MB Location JW-A-TW B M12-I-Car JS JW JW-I-Car C M11 M12-A-MB M12-A-BM M12-A-BM M12 M12-I-Car MQ QLS JS-I-Car M11-I-Sed

M11-A-BMM12-A-MBM11-I-BoulFM12-A-MB JW-I-Car M11-I-Sed M12-A-BM M11-I-Sed QLS-I-SedOMZ JS-I-Car A M11-A-NCB M11-I-Sed M11-I-Sed QLS-I-SedOMZ MQ-I-HCor QLS-I-SedOMZ E(D)

Figure 2-6: A. Cluster analysis and B. two-dimensional MDS ordination of meio-epifauna associated with natural rocks of Costa Rica cold seeps. In the dendrogram (A), the four clusters, produced by a threshold similarity of 65 % are shown, however SIMPROF test support five (A to E) main groups (black lines). In the ordination (B) green lines show four distinct groups (D nested Chapter 2 25 within E) separated by a threshold similarity of 65 %. A=Active, BM=Bacterial mats, BoulF=Boulder Field, Car=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco scar, JW= Jaco wall, M11=Mound 11, M12=Mound 12, MB=Mussel beds, MQ=Mound Quepos, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, Sed=Sediments, TW=Tubeworms.

Across all activities and habitats, the one-way ANOSIM analyses (Table 2-1, test 1) showed significant differences in higher taxa composition and abundance between locations. However, pairwise differences between locations show only a few being important (R close to 1 and p less than 1 %), reflecting differences between the clusters-groups separated in the clusters and MDS analyses. Across all locations and habitats, although greater number of taxa and densities and taxonomic groups were found in inactive sites than in active ones, according to the ANOSIM (Table 2-1, test 2) there were no significant differences in community composition and abundances between seepage activities. For habitats, only weak differences were found when comparing all locations and activities (ANOSIM Table 2-1, test 3). In this last analysis, substrates that were retrieved from inactive sites (on sediments) and had a background categorized as hard surfaces (rocks, corals, boulders) were not grouped in the six categories shown in Figure 2-3 (upper part of the figure); this was intended to establish whether the type of hard substrate found on the sediment could influence the clusters. This analysis also was done because when performing an ANOSIM test with the six established habitats, no significant differences were found, but when substrates were separated (i.e., inactive sediments and inactive rock types), differences were found (Table 2-1, test 3).

A simultaneous comparison was carried out between activities and habitats, using a 2-way nested ANOSIM (factor 1 activity, factor 2, habitats nested within activities). No significant differences were found between rocks located in active vs. inactive sites (R=-0.056, p=0.643). But a slight variation was found in habitats within seepage activity R=0.281, p=0.006) (Table 2-1, test 4).

Table 2-1: Results of the various ANOSIM analyses comparing composition and density of meio-epifaunal higher taxa between location, seepage activity, and habitat, for both natural carbonate rock substrates, and experimental (10.5 month) colonization rocks. A=Active, BM=Bacterial mats, BoulF=Boulder Field, Car=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco scar, JW=Jaco wall, M11=Mound 11, M12=Mound 12, MB=Mussel beds, MQ=Mound Quepos, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, Sed=Sediments, TW=Tubeworms. *= significant.

Sample statistic Permutation Significance level % (R) used NATURAL ROCKS ANOSIM test 1 (28 substrates) Global test Locations 0.436 9999 0.01 % (p=0.0001)* Pairwise tests JS vs. JW -0.167 10 80 JS vs. M11 0.539 45 6.7 JS vs. M12 0.188 78 21.8 JS vs. MQ 1 3 33.3 JS vs. QLS 1 10 10 JW vs. M11 0.788 165 0.6 26 Meio-epifauna Costa Rica

JW vs. M12 0.017 364 44.5 JW vs. MQ 0.333 4 50 JW vs. QLS 1 10 10 M11 vs. M12 0.238 9999 1.1 M11 vs. MQ 0.821 9 11.1 M11 vs. QLS 0.984 165 0.6 M12 vs. MQ 0.312 12 16.7 M12 vs. QLS 0.859 364 0.3 MQ vs. QLS 1 4 25 ANOSIM test 2 (28 substrates) Global test Seepage activity -0.009 49.4 % (p=0.494) ANOSIM test 3 (28 substrates) Global test Habitat 0.242 1 % (p=0.01)* Pairwise tests Car vs. TW 0.104 28 39.3 Car vs. BM -0.19 210 91 Car vs. NCB 0.067 7 57.1 Car vs. Sed 0.057 462 25.8 Car vs. BoulF -0.556 7 100 Car vs. MB 0.048 210 39 Car vs. HCor 0.311 7 42.9 Car vs. SedOMZ 0.938 84 1.2 TW vs. BM -0.071 15 60 TW vs. NCB 1 3 33.3 TW vs. Sed 0.427 28 10.7 TW vs. BoulF 1 3 33.3 TW vs. MB 0.429 15 13.3 TW vs. HCor 1 3 33.3 TW vs. SedOMZ 1 10 10 BM vs. NCB 0.25 5 60 BM vs. Sed 0.127 210 19.5 BM vs. BoulF -0.25 5 60 BM vs. MB -0.052 35 62.9 BM vs. HCor 0.167 5 60 BM vs. SedOMZ 0.667 35 2.9 NCB vs. Sed -0.356 7 85.7 NCB vs. MB 0.167 5 40 NCB vs. SedOMZ 1 4 25 Sed vs. BoulF -0.333 7 71.4 Sed vs. MB 0.206 210 8.6 Sed vs. HCor 0.244 7 28.6 Sed vs. SedOMZ 0.747 84 2.4 BoulF vs. MB -0.167 5 60 BoulF vs. SedOMZ 1 4 25 MB vs. HCor 0.583 5 40 MB vs. SedOMZ 1 35 2.9 HCor vs. SedOMZ 1 4 25 ANOSIM test 4 (28 substrates) Global test Two-way nested (B within A) - B(A). A=Activity, B=Habitat Chapter 2 27

Tests for differences between unordered Activity groups (using Habitat groups as -0.056 64.3 % (p=0.643) samples) Tests for differences between unordered 0.281 0.6 % (p=0.006)* Habitat groups (across all Activity groups) COLONIZATION EXPERIMENTS ROCKS (10.5 MONTHS) ANOSIM test 5 (11 substrates) Global test Seepage activity 0.144 15.4 % (p=0.154) ANOSIM test 6 (11 substrates) Global test Habitat 0.415 3.2 % (p=0.032)* MB vs. NMB 0.357 15 20 MB vs. TW 1 3 33.3 MB vs. RB 1 3 33.3 MB vs. Sed 1 3 33.3 NMB vs.TW 0.607 15 6.7 NMB vs. RB -0.214 15 73.3 NMB vs. Sed -0.25 5 80 TW vs. RB 1 3 33.3 TW vs. Sed 1 3 33.3 RB vs. Sed -1 3 100 ANOSIM test 7 (11 substrates) Global test Two-Way Nested (B within A) - B(A). A=Activity, B=Habitat Tests for differences between unordered Activity groups (using Habitat groups as 0.037 50 % (p=0.5) samples) Tests for differences between unordered 0.247 20.4 % (p=0.204) Habitat groups (across all Activity groups) ANOSIM test 8 (4 substrates) Global test NMB (Seepage activity) -0.25 100 % (p=1)

The SIMPER analysis showed Nematoda being the main taxon responsible for similarities between locations. Between habitats, the main taxa mainly are Nematoda and Copepoda (Table 2-2).

Table 2-2: Similarity results calculated by SIMPER (28 substrates). Taxa contributing most to the similarity between ANOSIM analysis (between location, and between habitat). Groups were ranked according to their average similarities (Av. sim.) and taxa according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given. Nematodes are the most important contributors to similarity in most groups. Groups MQ, NCB, BouldF, and HCor less than 2 substrates in group. A=Active, BM=Bacterial mats, BoulF=Boulder Field, Car=Carbonates, HCor=Hard Coral, I=Inactive, JS=Jaco scar, JW=Jaco wall, M11=Mound 11, M12=Mound 12, MB=Mussel beds, MQ=Mound Quepos, NCB=Near clam beds, OMZ=Oxygen Minimum Zone, QLS=Quepos landslide, Sed=Sediments, TW=Tubeworms. 28 Meio-epifauna Costa Rica

Between locations: Group JS Average similarity=75.57 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 1.6 18.2 24.1 24.1 Copepoda 1.3 12.3 16.3 40.1 Polychaeta 0.7 7.9 10.5 50.9 Bivalvia 0.5 5.9 7.8 58.7 Ostracoda 0.6 5.9 7.8 66.5 Gastropoda 0.5 5.7 7.6 74.1 Group JW Average similarity=64.0 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 1.1 13.1 5.5 20.5 20.5 Copepoda 1.2 12.5 5.1 19.6 40.1 Nauplii 0.9 7.8 5.2 12.1 52.3 Ostracoda 0.6 7.7 4.6 12.0 64.3 Polychaeta 0.7 5.4 6.4 8.4 72.7 Group M11 Average similarity=76.1 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 1.8 15.7 6.1 20.6 20.6 Copepoda 1.6 13.6 8.7 17.9 38.5 Others 1.0 8.2 6.4 10.8 49.3 Nauplii 0.8 6.9 5.7 9.1 58.5 Polychaeta 0.8 6.9 11.7 9.1 67.6 Ostracoda 0.6 4.9 9.1 6.4 74.0 Group M12 Average similarity=66.6 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 1.2 15.4 4.7 23.1 23.1 Copepoda 1.2 14.3 6.0 21.4 44.5 Others 0.7 7.5 1.9 11.2 55.8 Polychaeta 0.6 6.6 1.9 9.9 65.7 Nauplii 0.7 5.7 1.3 8.5 74.2 Group QLS Average similarity=69.8 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 2.5 35.0 7.3 50.1 50.1 Copepoda 0.7 11.3 4.7 16.2 66.3 Polychaeta 0.7 8.6 4.1 12.3 78.6 Between habitats: Group Car Average similarity=66.1 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Copepoda 1.3 13.9 6.8 21.0 21.0 Nematoda 1.3 13.2 4.9 20.0 41.0 Ostracoda 0.6 6.0 7.1 9.1 50.2 others 0.7 5.4 1.3 8.2 58.4 Chapter 2 29

Bivalvia 0.5 5.4 5.5 8.1 66.5 Gastropoda 0.6 5.3 7.1 8.0 74.5 Group TW Average similarity=67.2 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 1.0 14.2 21.1 21.1 Copepoda 0.9 13.7 20.3 41.4 Ostracoda 0.6 9.1 13.5 54.9 Nauplii 0.6 8.6 12.7 67.7 Polychaeta 0.5 6.5 9.6 77.3 Group BM Average similarity=65.7 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 1.6 18.5 7.7 28.1 28.1 Copepoda 1.1 14.5 8.0 22.1 50.2 Polychaeta 0.7 8.4 9.2 12.7 62.9 Nauplii 0.7 7.9 4.0 12.0 74.9 Group Sed Average similarity=67.9 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 1.5 13.6 10.9 20.0 20.0 Copepoda 1.4 11.8 5.4 17.4 37.4 Nauplii 0.8 6.9 6.2 10.2 47.6 Others 0.9 6.9 3.7 10.2 57.8 Polychaeta 0.7 6.1 5.4 9.1 66.9 Ostracoda 0.6 5.5 5.8 8.2 75.0 Group MB Average similarity=71.78 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Copepoda 1.4 14.8 7.9 20.7 20.7 Nematoda 1.3 13.9 4.7 19.4 40.1 others 0.9 11.4 9.8 15.9 56.0 Gastropoda 0.6 7.8 4.1 10.8 66.8 Polychaeta 0.6 7.7 7.6 10.8 77.6 Group SedOMZ Average similarity=69.8 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Nematoda 2.5 35.0 7.3 50.1 50.1 Copepoda 0.7 11.3 4.7 16.2 66.3 Polychaeta 0.7 8.6 4.1 12.3 78.6

A combination of environmental variables (depth, temperature, O2, and pH) and taxa abundances had a weak correlation (RELATE test, Rho=0.492, p=0.001). Table 2-3 shows the result of the best combination of the four environmental variables measured that explain the distribution of the assemblages found. The second column of the table shows Spearman´s Rho for the four highest combinations in each group. The depth and temperature showed the highest coefficient (Rho=0.507), 30 Meio-epifauna Costa Rica followed by depth (Rho=0.499); in other words, they are the variables that "best explain" the biological scheme found in the ordination.

Table 2-3: BEST-BIOENV results. Only the best four correlations are shown. Best result for each number of variables are in bold. * best result.

N° variables Rho Depth Temperature O2 pH 1 0.499 x 1 0.491 x 1 0.491 x 1 0.057 x 2 0.507* x x 2 0.483 x x 2 0.473 x x 2 0.440 x x 3 0.480 x x x 3 0.464 x x x 3 0.447 x x x 3 0.404 x x x 4 0.492 x x x x

Based on ANOSIM between locations and the observed differences in composition (through locations Fig. 2-4 A) a Kruskal-Wallis test was carried out on densities and number of species to further explore those differences. The test showed that there no significant difference between location medians H (chi2)=6.873; Hc (tie corrected)=7.348, p (same)=0.196.

2.3.3 Abundance and composition of experimentally deployed substrates

A total of 18,321 individuals were collected in the 11 substrates deployed at Mound 12 and recovered after 10.5 months of colonization (Appendix 4). Total mean meio-epifaunal density was 20.94±14.57 ind.10cm-2.

The meio-epifauna community after 10.5 months of colonization was composed of 14 phyla/subdivisons, including permanent and temporary meio-epifauna (Appendix 4). Copepoda, Nematoda, nauplii and Polychaeta were among the most representative groups (Fig. 2-7). In these experiments, nauplii larvae were found, in all samples (Appendix 4, Fig. 2-7). Permanent meiofauna accounted for 86.8 % of the total number of organisms, while temporary meio-epifauna represented the remaining 13.2 %. Seven percent of all copepods were ovated and nauplii larvae (7 %) were abundant. Inactive and active sites had similar mean number of taxa, being 7.8±2.2 taxa in inactive and 7.0±1.1 taxa in active sites.

Taking into account all experimental rock samples, the most prominent taxa were copepods, with variable densities ranging from 2.5 ind.10 cm-2 (Rock # 3 - Active, MB) to 36.4 ind.10 cm2 (Rock# 9- Active, NMB); they were followed by nematodes, with densities ranging between 0.02 ind.10 cm- 2 (Rock# 6 - Active, TW) to 18.5 ind.10 cm-2 (Rock# 10 - Active, NMB) (Appendix 4). Chapter 2 31

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Relative abundance abundance Relative (%) Polychaeta 20% Nauplii 10% Nematoda Copepoda 0% R#2-4-I-NMB R#7-8-I-RB R#11-I-Sed R#1-3-A-MB R#5-6-A-TW R#9-10-A-NMB

Figure 2-7: Relative abundance of meio-epifauna in experimental rocks after 10.5 months of colonization (ordered by habitat and seepage activity). Densities are averaged by habitat. Labels show the number (#) of each rock deployed in each habitat-activity combination; there was only one rock (R#11) in the sediment habitat. A=Active, I=Inactive, NMB=Near mussel bed, RB=Rubble, Sed=Sediments, TW=Tubeworms.

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Figure 2-8: Mean density (ind.10 cm-2±1 SD) in experimental rocks after 10.5 month of colonization (Mound 12), all rocks deployed in inactive sites (near mussel bed, in rubble and in sediments) combined, in comparison to active sites. A=Active, MB=Mussel bed, NMB=Near mussel bed, TW= Tubeworms. 32 Meio-epifauna Costa Rica

Averaging by habitat and seepage activity (Fig. 2-8) it was observed that rocks deployed in Near Mussel Beds (NMB, active) had the highest mean density of organisms (35.5±16.0 ind.10 cm-2) (here inactive corresponds only to rocks present in sediments and rubble, there is no variation of hard substrates as in natural rocks; that's the reason everything was averaged as a category called "inactive"). In NMB (active), copepods had high average density (20.1±23.0 ind.10 cm-2), followed by nematodes (10.1±11.9 ind.10 cm-2), and polychaetes (2.54±3.1 ind.10 cm-2); in fact, nematodes and polychaetes in NMB habitat had the highest mean densities in all habitats in Mound 12. The next higher mean density occurred in NMB (but inactive) with 29.1±19.2 ind.10 cm-2. Therein, copepods were the group with the highest mean density of all habitats on Mound 12 (22.7±17.8 ind.10 cm-2), while nematodes only had 4.7±0.7 ind.10 cm-2. The habitat Sediments (inactive) had a total density of 32.2 ind.10 cm-2, although it consisted of a single rock; the contribution of copepods was 28.4 ind.10 cm-2 and that of nematodes was 1.5 ind.10 cm-2 (Appendix 4).

As for natural substrates, cluster and multi-dimensional scaling (MDS) ordination of meio-epifaunal higher taxa colonizing experimental rocks (Fig. 2-9 A, B) show no consistent grouping or segregation according to habitat or seepage activity. In addition, SIMPROF showed all rocks to conform an homogeneous group. The only pattern observed is that the two rocks located in tubeworms (TW, active) and the two located in mussel beds (MB, active) have similar composition. All other rocks are grouped together, and one of the rocks (#2) deployed near a mussel bed (NMB, inactive) came out isolated (Fig. 2-9 A, B).

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Figure 2-9: A. Cluster analysis and B. two-dimensional MDS ordination of meio-epifauna associated with 11 experimental rocks of Costa Rica cold seeps after 10.5 months of colonization. In A., there is a single homogeneous group (red lines) from SIMPROF (the black broken line marks 65 % similarity level). In B., green lines group clusters produced in A. at the 65 % similarity threshold. A=Active, I=Inactive, NMB=Near mussel bed, RB=Rubble, Sed=Sediments, TW=Tubeworms. R#=sample number.

The SIMPER analysis also showed a single group (at average similarity of 75.3 %), with Copepoda being the main taxon responsible for similarities; its contribution was 31.4 % (Table 2-4).

As in natural rocks, greater mean densities and number of taxa were found in inactive vs. active sites (across all habitats), and the ANOSIM (Table 2-1, test 5) analysis also showed no significant differences in higher taxa community composition and abundance between seepage activities. Comparing mean density across habitats (combining activity) the same pattern of no differences was found as for natural rocks H (chi2)=1.801; Hc (tie corrected)=1.898, p(same)=0.7545, but the ANOSIM (Table 2-1, test 6) analysis did demonstrate significant differences between habitats. In the 2-way nested ANOSIM (habitat nested within activities), no significant differences were found for either factor (Table 2-1, test 7).

As for experimental rocks there was one habitat (near mussel beds - NMB) in which there were active and inactive sites, and a 1-way ANOSIM comparing activities was carried out (Table 2-1, test 8). No significant differences in higher taxa community composition and abundance between seepage activities at NMB habitat were shown. 34 Meio-epifauna Costa Rica

Comparing the densities of the two dominant taxonomic groups in natural and experimental rocks, important differences in nematodes and copepods densities were evident, while in other groups densities were similar (Fig. 2-10).

30

25

2 20

15

10 Mean Ind. m Ind. Mean

5

0 Nematoda Copepoda Others

Natural Experimental

Figure 2-10: Mean density ±1 SD (ind.10 cm-2) comparing main taxa between natural (blue) substrates and in experimental rocks (red) after 10.5 months of colonization. Others summarize all other taxa found different from the most representative groups.

Table 2-4: Results of the SIMPER analysis showing the higher taxa contributing most to the similarity of the single group of 11 experimental rocks. Groups were ranked according to their average similarities (Av. sim.) and taxa according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given.

Single group Average similarity=75.3 % Taxa Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Copepoda 1.8 23.7 6.6 31.4 31.4 Nauplii 1.0 14.4 5.8 19.2 50.5 Nematoda 1.2 13.8 2.9 18.4 69.0 Polychaeta 0.8 9.6 3.7 12.7 81.7

2.4 Discussion

Composition and densities. This work advances in characterizing the meio-epifaunal higher rank taxa associated with loose carbonate rocks in deep-water cold seeps, and provides insights into the initial (months) steps of colonization on denuded rocks. Our study highlights the large number of taxa found associated with natural carbonate substrates, with 27 in total, while only 14 taxa were found in experimental rock substrates after only 10.5 months of colonization. In contrast, in biogenic Chapter 2 35 substrates, such as tubeworm bushes and mussel beds in cold seeps of the Gulf of Mexico, the epizooic metazoan meiobenthic community was composed of a limited number of higher taxa, including only Nematoda, Copepoda, Ostracoda, Halacarida, and Kinorhyncha, and recorded in remarkably low abundances (Bright et al., 2010). As expected, nematodes and copepods were the most representative groups in our study. Based on several meiofaunal studies carried out in soft, hard and phytal substrates, it has been established that nematodes and copepods are dominant; these two groups are known to be the most successful in terms of density, biomass, and number of species (Donavaro & Fraschetti, 2002; Zaleha et al., 2010; Sarmento et al., 2012). Interestingly, in our study we found that on natural carbonate substrates nematodes dominated with 58.1 % (vs. 24.9 % Copepoda), and after 10.5 months of colonization copepods dominated with 69.0 % (vs. 17.6 % Nematoda). This results does not correspond to the patterns found in other studies. Comparatively, soft bottoms are largely dominated by nematodes, whereas hard bottoms are generally dominated by harpacticoid copepods, isopods and amphipods. Specifically, in biomass values, due to their exoskeletons, crustacean dominance on hard substrates becomes more evident (Donavaro & Fraschetti, 2002).

In seep sediment communities in particular, several authors established that metazoan meiofauna is composed largely of nematodes (Levin, 2005; Van Gaever et al., 2009; Vanreusel et al., 2010), with low species richness and elevated dominance, compared to more typical deep-sea sediment environments (Van Gaever et al., 2009). Nematodes are considered as important indicators of habitat heterogeneity in marine environments, because they are common, numerous and speciose, and in close contact with seafloor related processes (Vanreusel et al., 2010). They occur in a wide range of sometimes extreme habitats including microbial biofilms on hard substrates (Fonsêca-Genevois et al., 2006). In deep-sea sediments, they comprise 80-97 % of all benthic meiofauna (Pfannnkuche, 1985; Vanhove et al., 1995; Bik et al., 2010; Lampadariou et al., 2013). However, despite their numerical importance, little is known on their ecology and distribution in the deep sea, especially in association with seeps and vents (Vanreusel et al., 2010), and several substrates common to these habitats (e.g. rocks and sunken woods). Their importance in sedimentary habitats is explained by their morphological and ecological adaptations; most of them are long and slender organisms, thus able to move between the sand grains rather than to crawl over a large surface (Raes & Vanreusel, 2005). Other morphological adaptations to extreme conditions could exist; thiobiotic nematodes (present in sulfur-oxiding conditions) exhibit a more slender morphology than oxybiotic species (Jensen, 1987). However, Buck & Berry (1998) found no differences in the relationship of body length and body diameter between seeps and control samples. Nonetheless, some families show morphological adaptations to an epifaunal life strategy (Raes & Vanreusel, 2005). On deep sea, dead fragments of the coral Lophelia pertusa, predominance of nematodes belonging to the epifaunal families Epsilonematidae and Draconematidae was observed. These families are morphologically adapted to “walk” over hard substrates; they have a looper caterpillar-like locomotion using ventral setae (ambulatory setae: Epsilonematidae) or tubes (posterior adhesion tubes: Draconematidae) on their posterior body region, sometimes together with tubes on or near the head capsule (cephalic adhesion tubes: Draconematidae) (Raes & Vanreusel, 2005). Also, on shallow water corals, dominant species were “hard-body” nematodes able to withstand physical stress thanks to morphological adaptations such as stouter body shape, ornamented cuticle, cephalic capsule, somatic setae and developed spinneret plus caudal glands (Armenteros et al., 2012). In summary, nematodes 36 Meio-epifauna Costa Rica success in life in sediment seems to be closely related to their body-shape, but there are special adaptations to live on hard surfaces as well. It is thus possible that their dominance in seep carbonate substrates is a real phenomenon which so far had not been studied in detail.

On the other hand, the high presence of copepods is not uncommon, neither for shallow waters nor for deep water. The copepod dominance in the relatively short term duration of our experiment suggest these mobile metazoans are pioneers during early stages of succession on hard substrates of the deep sea. Nematodes, in contrast, seem to dominate the developed meio-epifaunal communityof the natural carbonate rocks in Costa Rican cold seeps. Although other studies to compare the dominance of copepods on cold seep hard substrates are unknown, on natural shallow environments, the first settlers were copepods, i.e. cyclopoids, dominating all sampled temporary ponds (Frisch & Green, 2007). The latter authors suggest that fast dispersal and dominance of certain cyclopoid copepods during early colonization is related to their ability to store sperm and rapid individual development. This could indicate that, at least in shallow waters, environmental conditions would not be determining colonization, but rather the group's own biological characteristics. Also, harpacticoids colonized experimental submerged shallow platforms within days (see Coull, 1988 review). In shallow areas, studying species assemblages of benthic harpacticoid copepods on tide rock-pool seaweeds it was found that harpacticoid copepods represented 46.1 % of total meiofauna in the sediment adjacent to Ulva reticulata while only 26.6 % was found inhabiting the seaweed (Zaleha et al., 2010). Fonsêca-Genevois et al. (2006) found, also for shallow waters but in bare aluminium surfaces, that harpacticoid copepods were the fastest meiofaunal colonizers, consistent with their comparatively high propensity to emerge from sediments and colonize a variety of artificial substrates. Although in such experiments the first nematodes appeared on the plates after two days, they reached maximum densities later than did copepod populations. Thus, it is not unlikely that copepods also are the first colonizers in the deep sea. In chapters 4 and 5, we present the results of a more thorough study on copepods and nematodes respectively.

For the findings of nematode dominance in natural rocks and the initial dominance of copepods in 10.5 months of colonization, another factor to consider is that changes in spatial and temporal distributions of organisms may be due to natural fluctuation in recruitment, physical perturbation or pollution effects (or a combination of these) (Gobin, 2007). A study in hydrothermal vent mussel beds on the northern and southern East Pacific Rise, looking for the influence of mussel bed age on meiofaunal assemblages, found that nematodes were the numerically dominant meiofaunal taxon at the majority of sites, but copepods were dominant at youngest mussel beds stations (Copley et al., 2007). These results may suggest that community composition changes with the residence time of the substrate. At this point, we cannot assert whether any of these effects had played a role in this difference between natural and experimental substrates.

Seepage activity. Recently, Levin et al. (2015), in the same study area of Costa Rica, described the macrofaunal communities on carbonate substrates between 400 to 1850 m in depth, establishing that species diversity is higher on rocks exposed to active seepage, with groups like gastropods and polychaetes being dominant, whilst others like crustaceans, cnidarians, and ophiuroids were better represented on rocks at inactive sites. The above may be indicating preferences or limitations of the various taxonomic groups for the different environmental conditions resulting from seepage activity. In our case, the highest densities of meio-epifauna were always associated with inactive sites. In Chapter 2 37 quantitative comparisons of seep-active bacterial mats with non-seep control sites, it was found that nematodes are the dominant biomass contributors of the seep-active samples (Buck & Barry, 1998). Our results indicate that in average density, Nematoda was dominant in rocks located in active and inactive sites, exceeding copepods in both cases. In experimental rocks, both in active and inactive sites, copepods were dominant. The seepage activity is a factor that needs to be characterized in situ. According to Case et al. (2015) seepage flux can increase and decrease, as well as shift spatially, on a scale of days to weeks to centuries. The task to establish how these changes can affect meio- epifaunal colonization and succession processes in time (short or long) is yet to be undertaken.

Habitat. The density and higher taxonomic composition of meio-epifauna associated with the several habitats studied here showed differences. It is expected that the three-dimensional structures that are created on the bottom (whether of organic or inorganic origin) will add complexity to the habitat and generate microhabitats that favor the diversity of the organisms and groups found. For metazoan meio-epifauna associated with cold water corals, it has been found that the presence of large biogenic structures on the sea floor favors harpacticoid copepods, naupliar larvae and polychaetes (Raes & Vanreusel, 2005). Whereas at hydrothermal vents, it has been considered that a high structural complexity in the epifaunal thickets results in a rich meio-epifaunal life; that is, tightly interwoven tubes are more densely populated than bush-like structures (Giere, 2009). A study of epizooic meiobenthos associated with tubeworm and mussel aggregations in the northern Gulf of Mexico cold seeps, found that in all tubeworm samples from different locations the most prominent taxa were the nematodes (relative abundances between 57 % and 90 %), followed by the copepods (10-43 %), and the relative distribution of higher taxa was more variable in mussel bed samples. In three samples, nematodes dominated (66-82 %), followed by copepods (17-30 %), and in two samples copepods were most abundant (82 % and 99 %) (Bright et al., 2010). Levin et al. (2015), stated that at a coarse taxonomic level, the composition of carbonate macrofauna in active Costa Rica seep sites bears remarkable resemblance to the biota of temperate and tropical rocky intertidal shorelines. In shallow waters, on rocky substrates, macroalgae generally increase substrate complexity, offering refuges from predation; and increase habitat diversity, enhancing substrate colonization (Donavaro & Franchetti, 2002). These same reasons could be applied to deep-water meio-epifauna. Foundation species influence the abundance, composition, and structure of the associated community and can provide food resources, living space, favorable settlement conditions, a refuge from predators, and/or from environmental stress (Bright et al., 2010). However, in our results we do not observe a clear pattern regarding the structural complexity of the habitat. Natural rock substrates located on sediments or in other types of habitats (with foundations species) did not show clear patterns in mean number of taxa or densities. These relationships could be explored at lower taxonomic levels.

Oxygen Minimum Zone. The only OMZ site of this study (Quepos landslide location) was where the highest densities of nematodes were found. It has been documented that many nematode species have revealed remarkable abilities to withstand stress and adverse conditions such as living under low-oxygenated conditions, including anoxic and sulphidic sediments, compared to other meiobenthic taxa (Levin et al., 2001; Giere 2009; Sandulli et al., 2015), which could explain this taxon’s high density therein. However, in this location the mean number of total taxa compared to the other locations, was the lowest (i.e., 14 in Jaco Summit, 13 in Mound 11, 11 in Jaco wall and Mound Quepos, 10 in Mound 12, and 8 in Quepos landslide). This supports what was argued by 38 Meio-epifauna Costa Rica

Neira et al. (2001), that the absence of many groups at OMZ sites suggests that oxygen deficiency may have an influence on the meiofaunal composition at higher taxonomic levels. This is also the case for copepods, a group that apparently does not have these broad tolerances to extreme conditions, which also had lower densities at Quepos landslide location. Harpacticoids are known to have a low tolerance to decreased oxygen levels and are reported to occur in the upper oxygenated bottom layer (several authors in Urkmez et al., 2015). This may indicate that although harpacticoid species assemblage increases in number and diversity with increasing micro-spatial habitat complexity (Zaleha et al., 2010) there could be a more significant limiting factor such as oxygen level. Moreover, meiofaunal studies in OMZ have revealed a high nematode abundances at low oxygen concentrations <0.02 mL.L-1, supporting the hypothesis that densities are enhanced by an indirect positive effect of low oxygen involving reduction of predators and competitors, and preservation of organic matter leading to high food quality and availability (Neira et al., 2001). It was also found that both seeps and OMZ generate distinct and unique nematode assemblages, although they share certain environmental and faunal characteristics worldwide (Guilini et al., 2012; see also chapter 5).

Other environmental variables. Although there are some known patterns related to environmental variables such as O2, in our study depth and temperature are the variables that best explain the biological scheme found with natural rocks. However, this information was not obtained by habitat. Copepods are known to be restricted to the oxygenated regions in sediments. In that environment, they tend to be found on or just beneath the surface of muds (Wells, 1988). This fact is reaffirmed by Wetzel et al. (2001) who explained that vertically in muddy sediments, total meiofaunal abundance typically declines with increasing sediment depth through the upper 4 - 6 cm. But on a fine scale, however, harpacticoids are the most abundant meiofaunal taxon in the upper 4 mm, while many nematodes species have deeper abundance maxima. In this sense, we found the highest mean density of copepods occurred in the inactive areas, which could demonstrate their preference for better oxygenated places; for cold-seep sediments are considered extreme environments where the associated fauna is exposed to high levels of toxic hydrogen sulfide, high concentrations of methane and low oxygen concentrations (Sibuet & Olu, 1998; Levin, 2005; Cunha et al., 2013). Powell et al (1986) at brine seeps in the Gulf of Mexico found that outside the influence of the seep, meiofaunal copepods predominate. They determined that nematodes exploit sulfide systems considerably better than copepods (as an increase in sulfide concentration will increase the relative importance of nematodes), which disagrees with results found for shallow-water nematodes associated with mangroves (see Laurent et al., 2013). Hence, much of the significance of the nematode/copepod ratio can be reinterpreted based on the relative importance of sulfide-tolerant organisms in the community. Therefore, future studies must take into account the chemical variations presented in each habitat (but not in each location), as these may be what are actually structuring the community.

Colonization. The colonization processes for meio-epifaunal organisms in deep waters are not yet clear. How do nematodes colonize autogenic rock substrates in Costa Rica cold seeps? This is another question that needs to be answered. Although some theories are proposed, it is important to understand how these organisms begin this process of colonization. Nematodes like other meiofauna, have very limited active dispersal capabilities. They have no pelagic stages, although they can temporarily survive when suspended in water. Resuspension and passive transport by water currents may be important mechanisms by which they recruit (permanently or temporarily) onto submerged Chapter 2 39 substrates, but it is unclear whether and how nematodes maintain populations on such substrates (Fonsêca-Genevois et al., 2006). In our study area, Grupe (2014) found that macrofauna colonized experimental wood and carbonate substrates at higher densities in sites of seep fluid flow than in inactive sites (but at active seeps, carbonates contained significantly higher densities of colonizing macrofauna than did wood). In relation to what is determining the dominance of certain groups over substrates, Zaleha et al. (2010) documented in marine seaweeds, that the dominance of particular species of (harpacticoids) copepods is determined by the morphological features of the vegetation, which is closely related to the complexity of the substrate. However, this cannot be applied to our rock substrates, as they did not have the very noticeable roughness or 3D complexity typical of plants. However, factors such as small-scale porosity or roughness become important for small organisms such as meio-epifauna, where they find some form of protection and nourishment. Porosity and roughness could also condition the presence or absence of organisms adapted to live in non-interstitial environments. For example, for harpacticoid copepods it has been found that with an increase of roughness (measured as surface area), different types of harpacticoids might be found with various body shapes and leg characteristics that serve for clinging on surfaces (Zaleha et al., 2010). As in this study we did not measure roughness or porosity, so we cannot conclude if these factors have a role in the patterns found for both natural or experimental substrates. Nevertheless, although our rocks were generally roughened, smooth surfaces have been shown to have similar colonization processes (see in introduction Molecular fouling, Microfouling, and Macrofouling). For example, aluminium structures have been reported readily fouled by micro-organisms (Fonsêca- Genenois et al., 2006), which corresponds to the second colonization level of hard substrates, after macromolecular adhesion. What needs to be established is what kind of microorganisms. To better understand the colonization processes of meio-epifauna, it would be important to understand what happens at the first level of colonization. The meio-epifauna is in the second level, but the first is still uncertain.

Appendix 1. Locations, habitat, survey dates, and depths of substrates collection (natural rocks) in 2009 during AT 15-44 cruise by the Alvin manned submersible at Costa Rica. Act.=Seepage activity, A=Active, I=Inactive, OMZ=Oxygen Minimum Zone.

Location Alvin Habitat Act. Substratu Date Depth Latitude N Longitude W T O2 pH dive m number recovered (m) (°C) (mL.L.1)

Jaco summit 4510 Sediments I L2 Feb-03-09 742 9° 10.35673’ 84° 47.91574’ 6 0.38 -

4510 Sediments I S1 Feb-03-09 741 9° 10.35728’ 84° 47.91247’ 6 0.38 -

Jaco wall 4509 Tubeworms A S2 Mar-02-09 1789 9° 7.12873’ 84° 50.54744 2.69 1.89 7.76

4509 Carbonates I S3 Mar-02-09 1854 9° 7.00938’ 84° 50.51579’ 2.69 1.89 7.76

4509 Carbonates I L2 Mar-02-09 1856 9° 7.00938’ 84° 50.51579’ 2.69 1.89 7.76

Mound 11 4504 Bacterial mat A S2 Feb-25-09 1010 8° 54.84256’ 84° 18.22426’ 4.19 1.1-1.3 7.7

4505 Near clam bed A L2 Feb-26-09 1025 8° 54.84256’ 84° 18.22426’ 4.19 1.1-1.3 7.7

4504 Sediments I S3 Feb-25-09 1007 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

4504 Sediments I S4 Feb-25-09 1009 8° 55.35470’ 84° 18.23789’ 4.19 1.1-1.3 7.7

4504 Sediments I L1 Feb-25-09 1000 8° 55.40516’ 84° 18.24007’ 4.19 1.1-1.3 7.7

4505 Boulder field I S2 Feb-25-09 1020 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

4505 Sediments I S3 Feb-26-09 1019 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

4505 Sediments I S4 Feb-26-09 1019 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

Mound 12 4501 Mussel bed A S4 Feb-22-09 997 8° 55.78710’ 84° 18.77827’ 5.11 0.99-1.6 7.7

4502 Bacterial mat A S4 Feb-23-09 989 8° 55.77625’ 84° 18.77227’ 5.11 0.99-1.6 7.7

4502 Mussel bed A L2 Feb-23-09 987 8° 55.78601’ 84° 18.64849’ 5.11 0.99-1.6 7.7

4503 Mussel bed A S2 Feb-24-09 990 8° 55.79632’ 84° 18.74228’ 5.11 0.99-1.6 7.7

4503 Tubeworms A L3 Feb-24-09 990 8° 55.79632’ 84° 18.63813’ 5.11 0.99-1.6 7.7

Chapter 2 41

4511 Mussel bed A S1 Mar-05-09 997 8° 55.83972’ 84° 18.75155’ 5.11 0.99-1.6 7.7

4511 Bacterial mat A L1 Mar-05-09 996 8° 55.77733’ 84° 18.77173’ 5.11 0.99-1.6 7.7

4511 Bacterial mat A L2 Mar-05-09 998 8° 55.78764’ 84° 18.64849’ 5.11 0.99-1.6 7.7

4502 Carbonates I S1 Feb-23-09 994 8° 55.78601’ 84° 18.74119’ 5.11 0.99-1.6 7.7

4502 Carbonates I S3 Feb-26-09 987 8° 55.79632’ 84° 18.62723’ 5.11 0.99-1.6 7.7

4503 Sediments I S1 Feb-24-09 967 8° 55.78656’ 84° 18.74174’ 5.11 0.99-1.6 7.7

Mound 4506 Hard Coral I L2 Feb-27-09 1033 8° 58.09673’ 84° 37.95542’ 4.14 1.4-1.5 7.8 Quepos

Quepos 4512 Sediments-OMZ I S2 Mar-05-09 380 9° 12 84° 30 9.5/9.6 0.26 7.7 landslide

4512 Sediments-OMZ I S3 Mar-05-09 344 9° 12 84° 30 9.5/9.6 0.26 7.7

4512 Sediments-OMZ I L2 Mar-05-09 376 9° 12 84° 30 9.5/9.6 0.26 7.7

42 Meio-epifauna Costa Rica

Appendix 2. Habitat, survey dates, and depths of substrates deployed in 2009. A manned submersible recovered experimental substrates after ~317 days of deployment at Mound 12, Costa Rica. Act.=Seepage activity, A=Active, I=Inactive, R#= rock number.

Alvin dive Habitat Act. Substratum Date Alvin dive Date Depth Latitude N Longitude W deploy type and retrieve recovered (m) number deployed

4501 Mussel bed A R#1, 3 Feb-22-09 4586 Jan-07-10 997 8° 55.83538’ 84° 18.75591’

4501 Near mussel bed I R#2, 4 Feb-22-09 4586 Jan-07-10 997 8° 55.83484’ 84° 18.75482’

4502 Tubeworms A R#5, 6 Feb-23-09 4588 Jan-09-10 996 8° 55.78981’ 84° 18.74010’

4502 Rubble I R#7, 8 Feb-23-09 4588-89 Jan-09-10-10 997 8° 55.79524’ 84° 18.75482’

4511 Near mussel bed A R#9, 10 Mar-05-09 4589 Jan-10-10 997 8° 55.83376’ 84° 18.77118’

4511 Sediments I R#11 Mar-05-09 4587 Jan-08-10 1001 8° 55.85926’ 84° 18.77336’

3. Meio-epifaunal wood colonization in the vicinity of methane seeps

Abstract

In deep-sea environments, plant remains of several origins are found, including branches, twigs, leaves, and wood pieces, among others. As most of the deep-sea bottoms are oligotrophic and nutrient limited, plant remains provide an oasis of localized organic enrichment and a substrate for colonization. Sunken wood was suggested to play an important evolutionary role in the diversification of chemosynthetic ecosystems, possibly representing stepping stones for the colonization between vent and seep ecosystems. In order to understand early colonization processes of the Pacific Costa Rican meio-epifaunal, non-sessile assemblages associated with sunken wood, a field experiment was conducted on Mound 12 (8° 55.77842’ N, 84° 18.73083’ W) at ~1000 m water depth. Wood blocks were placed in four different habitats (Mussel beds, tubeworms, near mussel beds, rubble), and different local environmental conditions (seepage-active and seepage-inactive sites). Seven experimental Douglas fir wood blocks (each 1047.1 cm2 in surface area) were deployed from the R/V Atlantis using the manned submersible Alvin in February 2009 and recovered after 10.5 months in January 2010. Sample processing and analyses led to a data set of abundance (total 9,951 individuals) and spatial distribution of nine meio-epifaunal higher taxa/groups. Meio-epifaunal densities on individual wood blocks ranged from 3 to 26 ind.10 cm2. Copepods accounted for the highest abundances (75 %), followed by nauplii larvae (12 %) and nematodes (10 %). The maximum number of individuals (26.3 ind.10 cm-2) was found in blocks placed in seepage inactive areas (near active mussel beds) in contrast to 2.9 ind.10cm-2 in active areas (within a mussel beds). A hierarchical cluster analysis grouped blocks according to seepage activity and not to habitat, but tests of similarity showed no significant differences in higher taxon composition and abundances, probably owing either to substrate homogeneity or low sample size. Similarly to parallel colonization experiments carried out on carbonate rocks, copepods were the most abundant representatives, suggesting that this group is one of the most successful in colonizing in the early stage of succession, in this case while hard wood substrates are not yet decomposed or bored by bivalves. Future colonization studies should involve varied and longer time intervals, the use of several types of wood, and the effect of decomposition sulfides on colonisation to confirm the settlement patterns found.

Key words: Cold seeps, organic substrates, experiments, wood blocks, meio-epifauna, Pacific Ocean

50 Meio-epifauna Costa Rica

3.1 Introduction

When trying to understand the types and diversity of organisms associated to extreme deep-sea environments, a key process is their colonization. What are the main factors that intervene or control their colonization, whether on hard surfaces or in the sediments? On the deep-sea floor, a diversity of hard substrates are found, both of organic and inorganic origin, i.e., sunken wood, animal bones, authigenic carbonates, cold water corals, mollusk shells and polychaete tubes, among others. These substrates could be favorable for the development of small-sized meiofaunal organisms, which at the same time support larger-sized macrofaunal communities, following an elaborate trophic pattern.

The presence of organic remains in the deep sea such as animal carcasses, sunken wood, and sea- grass blades is common and was reported as early as 1895 (see review in Wolff, 1979). Many records of plant material are from basins in the vicinity of land and off large river mouths. Plant remains even occur frequently in deep-sea trenches (Gallo et al., 2015). Another fraction of such material is dispersed, especially during hurricanes, by rafting or by currents (Wolff, 1979; Amon, 2013). Comparatively, the different substrates (rocks, different types of wood, biogenic materials), have different levels of persistence and their effect on the deep-sea benthos thus differ. That is:

▪ Carbonate substrates and other rocks: long term persistence. ▪ Wood: intermediate term (as will be explained below). ▪ Animal remains: short term, as they are rapidly consumed by motile scavengers (Maddocks & Steineck, 1987).

In deep-sea environments, plant remains are abundant in many areas. Likewise, the origins of such plant remains include terrestrial, shallow-water, and epipelagic sources. Such debris occurs as branches, twigs, leaves, wood pieces, bark, fruits, seeds (e.g., coconut), and sea-grass blades (Wolff, 1979; Samadi et al., 2010). Chronologically, it is known that woody plants evolved in the Late Devonian, and since then they became a source of food in the deep sea, and wood-fall ecosystems like those of the present, had evolved at least by late Eocene time (Kiel & Goedert, 2006). Such natural wood substrate is colonized by deep-sea organisms, from mega, to macro, meio, and microfauna, although colonization can be variable in space and time (Tyler et al., 2007). Plant debris materials and their accumulation are characterized by their small areal extent, short residence time, scattered spatial distribution and seasonal occurrence (Maddocks & Steineck, 1987).

Most of the deep-sea bottoms are oligotrophic and nutrient limited (Palacios et al., 2006). Therefore the presence of plant remains provide a significant oasis of localized organic enrichment (Maddocks & Steineck, 1987; Amon, 2013). Unlike other types of sunken materials (e.g., whale remains) the spatial distribution of the sunken plant material is unknown. Remarkably, some studies suggest that the amount of woody debris and transfer rate from the land to the marine environment is increasing. The latter is due to the increase in frequency of extreme climate events which have increased the mobilization of such debris (Yücel et al., 2012). This increase could have negative consequences on the marine environments, for example, by providing substratee, transport and dispersion of non- natives species across the oceans. Understanding the processes of succession and decomposition occurring in these deep-marine substrates can be a difficult task. For this reason, the purpose of this Chapter 3 51 study was the experimental deployment of wood into deeper waters to allow the monitoring and study of early colonization and successional processes.

Despite growing interest in the study of deep-sea ecosystems, many biological and ecological questions remain about the linking role of their associated fauna. Most of the contributions have been based on macrofaunal communities, leaving out one of the most critical components in connecting trophic webs and processes that are presented there: the meiofaunal communities. Meiofauna can be found either as mud-dwelling infauna or hard substrate epifauna (or meio-epifauna) (Raes & Vanreusel, 2005). There are studies focusing on the soft-bottom meio-infauna, but the meio-epifauna is still poorly studied. According to Fonsêca-Genevois et al. (2006), and our own studies (chapters 2 to 5), meio-epifaunal community composition on hard substrates in the deep sea tends to differ radically from those in neighboring sediments; although their dynamics of colonization and succession in fouling processes remain almost unknown.

The meiofauna is defined by size, including the portion of the benthic community that will pass through a 1-mm sieve and be retained on a 32-µm sieve (Bright et al., 2010; Vanreusel et al., 2010). This size group of organisms has been less documented and studied than their larger invertebrate counterparts, even though they are extensively represented in the deep sea. Meio-epifauna can colonize a variety of surfaces, including large pieces of biogenic debris, sea-grasses, macro-algae, coral fragments, sponge skeletons, tubeworms, clam shells, manganese nodules or pebbles (Raes & Vanreusel, 2005), and authigenic carbonates (chapter 2). Associated fauna that inhabit plant material or sunken wood can use it as a substrate or shelter, feed on xylophagous microbes or act as predators or scavengers. There are also chemotrophic species that host or graze bacteria that utilize sulfides resulting from the decay of the wood (Wolff, 1979; Kiel et al., 2008).

Chemosynthetic communities are found in association with hydrothermal vents, cold seeps, whale carcases and wood fall ecosystems (Tunnicliffe et al., 2003). These systems are fueled largely by chemical energy, mainly methane and sulfide, which generate a high biomass of chemosynthetic organisms. Sunken wood ecosystems represent a source of carbon and energy for any heterotrophic organism able to consume or decompose plant material, and the electron receptors associated with sulfide released through decay, fuel the associated chemosynthetic microbes (Bessett et al., 2014). In addition, sunken wood seems to have played an important evolutionary role in the adaptation, dispersal and diversification of chemosynthetic ecosystems, representing a stepping stone for dispersal and colonization of organisms between vent and seep habitats (Distel et al., 2000; Palacios et al., 2006; Bessett et al., 2014; Plum et al., 2015). Studies show shared and related species in hydrothermal vents, cold seeps, and large organic falls (whale carcasses and wood) (Distel et al., 2000; Laurent et al., 2013; Plum et al., 2015).

Sunken woods are a type of cold seep ecosystem. As an extreme ecosystem, sunken woods show high amounts of organic matter and high concentrations of sulfur compounds (Laurent et al., 2013). Sunken woods are a temporary ecosystem, and their meiofauna is present in low abundance, with few dominant species; in some seeps, these woods are dominated by one or few nematode species (Zeppilli et al., 2018). However, very few studies have been carried out in cold seep areas in general and in sunken woods in particular, and these trends have not been fully corroborated. 52 Meio-epifauna Costa Rica

Most studies carried out with organic substrates attempted to understand colonization and community development. These studies were based on either collection of natural sunken wood or colonization experiments by deploying wood blocks (e.g., pine, Douglas fir) or other substrates like alfalfa. Other works also have focused on the identification of naturally sunken wood material (see Turner, 1977; Wolff, 1979; Maddocks & Steineck, 1987; Pailleret et al., 2007; Lorion et al., 2009; Bernardino et al., 2010; Samadi et al., 2010; Schander et al., 2010; Gaudron et al., 2010; Ockelmann & Dinesen, 2011; Bienhold et al., 2013; Bessett et al., 2014; Cunha et al., 2013; Cuvelier et al., 2014).

In Costa Rica wood from the upland forest is deposited on the slope, often in the proximity of numerous methane seeps. To better understand the meio-epifaunal community assemblages in cold- seep hard bottoms of the deep sea, and how their presence might vary among several habitats and reducing seepage activity, in an unexplored area of the Pacific, this research investigated the community structure, abundance, and the influence of habitats on meio-epifauna colonizing deployed wood blocks. Also, in order to explore their diversity and faunistic composition, a review of studies on the meiofauna and macrofauna associated with wood substrates and with other reducing environments (vents and seeps) is provided.

3.2 Material and Methods

3.2.1 Costa Rica Marine Setting and Mound 12

In the eastern Pacific, the Costa Rican continental margin has heterogeneous areas with different combinations of reducing systems, i.e., cold seeps and the recently reported hydrothermal seep by Levin et al. (2012). The Pacific continental margin off Central America shows a large number of cold seeps associated with faults, slump scars of submarine landslides, and mounds (Buerk et al., 2010). Off Costa Rica and southern Nicaragua, more than 100 seep localities have been identified (on average one seep every 4 km) (Sahling et al., 2008). Potential fluid seepage has been documented at 161 sites on the shelf and slope regions of Costa Rica (Kluesner et al., 2013).

3.2.2 Study area

At the tectonically convergent margin of Costa Rica, one of the best documented geomorphic features is Mound 12, located to the southeast of the Nicoya Peninsula (Fig. 3-1). Mound 12 is an active mud volcano, found at 1020 m in depth (Niemann et al., 2013) with diameters of about 1 to 1.6 km (Mau et al., 2006). Diapirism and mudflows have formed a roundish cone-shaped relief with an irregular pinnacle (NE) and a lower profile ridge in the SW (Niemann et al., 2013). The Mound is characterized by the presence of high methane concentrations in the water column, authigenic carbonates, and chemosynthetic communities (Crutchley et al., 2014). The mound seems to be most seep active at its pinnacle and the SW flank, characterized by microbial mats and other chemosynthetic organisms (mytilid mussels and Lamellibrachia siboglinid polychaetes) (Niemann et al., 2013). According to Mau et al. (2006), at Mound 12 bacterial mats are the dominant communities, with an occupied area of 1,500-5,000 m2. Chapter 3 53

The Costa Rican margin exhibits strong vertical hydrographic gradients (Levin et al., 2015).

Temperature ranges from 9.5 to 2.7 °C at 400 m - 1800 m depth, bottom-water O2 concentration ranges from 0.04 to 1.6 mL.L-1, and pH ranges from 7.7 to 7.8 (Levin et al., 2015). The output of methane at Mound 12 is 15.5-52.5x103 mol.yr-1, lower than that reported in other seas such as the Norwegian and the Mediterranean (Burkett, 2011).

Primary information for this study was obtained during two research expeditions performed by the Scripps Institution of Oceanography – U. C. San Diego, onboard R/V Atlantis and using the manned submersible Alvin. On the first expedition, cruise AT 15-44 (22-23 February 2009), wood blocks for colonization experiments were deployed. During the second expedition, cruise AT 15-59 (07-10 January 2010, 10.5 months later), the blocks were retrieved (Fig. 3-1).

3.2.3 Habitats selection

Four habitats were selected for experimental deployment of wood blocks. These were on mussel beds, near mussel beds, on tubeworm bushes, and on rubble. Mussel beds and tubeworm aggregations were considered to experience active methane seepage, and near mussel beds and rubble habitats were considered inactive (Table 3-1). Thus, habitats were nested within seepage activity in this experimental design, with two blocks deployed per habitat type, for a total of eight blocks. Activity was assessed visually from the submersible by the presence of seepage, indicated by bubbling, bacterial mats, and chemosynthetic invertebrate communities. The depth of the experiments was about 1000 m. All samples were obtained under a collecting permit issued by the Costa Rica Ministerio de Ambiente y Energía, Sistema Nacional de Áreas de Conservación (Levin et al., 2015).

Table 3-1: Sites on Mound 12 and related information of experimental wood blocks deployed on February 2009 and recovered after ~317 days by the manned submersible Alvin. Overall values of environmental variables measured in Mound 12 were: Temperature 5.1 °C, Oxygen (Winkler) 0.9-1.6 L-1, pH 7.6-7.7 (Levin et al., 2015). Act.=Seepage activity, A=Active, D=Depth, I=Inactive, ST&N= Substrate type and number. Wood #3 was lost.

Alvin Habitat ST&N Act. Date Date D Latitude N Longitude W dive deployed recovered (m) 4587 Mussel beds Wood #1 A Feb-22-09 Jan-08-10 997 8° 55.83538’ 84° 18.75591’ 4586 Mussel beds Wood #4 A Feb-22-09 Jan-07-10 997 8° 55.83538’ 84° 18.75591’ 4588 Tubeworms Wood #5 A Feb-23-09 Jan-09-10 995 8° 55.78981’ 84° 18.74010’ 4588 Tubeworms Wood #6 A Feb-23-09 Jan-09-10 995 8° 55.78981’ 84° 18.74010’ 4586 Near mussel Wood #2 I Feb-22-09 Jan-07-10 997 8°55.83484’ 84° 18.75482’ beds 4588 Rubble Wood #7 I Feb-23-09 Jan-09-10 997 8° 55.79524’ 84° 18.75482’ 4589 Rubble Wood #8 I Feb-23-09 Jan-10-10 997 8° 55.79524’ 84° 18.75482’

3.2.4 Colonization experiments on Mound 12

This current experimental wood-substrate project is part of a larger study by Scripps Institution of Oceanography, conducted to address metacommunity questions about macrofaunal linkages between carbonate, wood and organic substrates, as well as controls on macrofaunal communities in several 54 Meio-epifauna Costa Rica geomorphic settings on the Costa Rican margin (Grupe, 2014; Levin et al., 2015). In order to solve questions at different scales, only a subset corresponding to Mound 12 was analyzed in this study. The goal was to understand colonization on organic substrates as an approximation to natural sunken wood, and to describe the initial process, in this case of meio-epifauna, to complement colonization studies of authigenic carbonate rocks (Chapter 2).

Untreated Douglas fir wood (Pseudotsuga menziesii) was selected for the experiments, as representative of sunken wood that often reaches reducing ecosystems. Also, because this wood type has been used in other international experiments (e.g., Gaudron et al., 2010; Bienhold et al., 2013; Cunha et al., 2013); thus, its use in this study allows for cross-experiment comparisons.

Following Grupe (2014) for macrofauna, the wood was cut into blocks of approximately 9 x 9 x 24.6 cm, each with a total surface area of 1,047.1 cm2. Of the eight wood blocks deployed, one was lost. Each block was bagged in 1.6 cm polypropylene mesh, and a floating polypropylene loop was attached to aid in handling (Fig. 3-1). Lead weights were attached to the wood to ensure they were negatively buoyant. Blocks were deployed using the manned submersible Alvin. At retrieval, blocks were placed in an insulated biobox with Plexiglas compartments that maintained separation of fauna during recovery.

Abundance quantification. On board, samples were washed with filtered sea-water, and the liquid residual was sieved through 300 and 43 µm meshes to recover the meio-epifauna. From this process, mixtures of sediment, particulate organic matter, and specimens were obtained. The meio-epifauna fraction was fixed in 8 % buffered formalin. To separate specimens from sediment, Rose Bengal was added to formalin and the sample fraction retained by the 43-µm mesh net was added to Ludox HS 40 (density 1.3 g cm-3) for density decantation extraction (5-6 times) (following D. Leduc- Meioscool, 2013). All metazoan organisms were manually sorted, counted and identified to a higher taxonomic rank following Higgins & Thiel (1988) and other sources. A ZEISS Discovery.V8 stereo- microscope was used.

Similarity among blocks. Meio-epifauna was identified to the higher taxonomic level. Taxon density for each block was calculated. Density was standardized to individuals in 10 cm-2 of surface area, for comparison purposes.

To explore differences and similarities of higher taxa composition and abundance between activities and between habitats within activities, a CLUSTER and an MDS analyses were performed from a Bray Curtis dissimilarity matrix, using fourth root transformed data to down-weight high abundance groups. To look for statistically significant evidence of structure in samples, a SIMPROF test (Type 1, Significance level 5 %) was performed.

Chapter 3 55

#1 #2

#4

#5

#6

#8 #7

Figure 3-1: Map of the study area showing the location were wood blocks were deployed in Mound 12, Costa Rica. A-MB (active, mussel bed)=location of Woods #1 and #4; I-NMB (inactive, near mussel bed)=Wood #2; A-TW (active, tubeworms)=Woods #5 and #6; I-RB (inactive, rubble bottom)=Woods #7 and #8. Each wood block is illustrated just at recovery (Wood #3, located in an inactive site near mussel bed habitat was lost). Wood pictures by permission from Greg Rouse, Scripps Institution of Oceanography.

To determine the possible significant difference in meio-epifauna colonization between seepage activity and between habitats within activity a two-way nested Analysis of similarity (ANOSIM), with 9999 permutations, and a p<0.05 significance level was constructed. This tested two 56 Meio-epifauna Costa Rica hypotheses: There was no differences in the composition and density of meio-epifauna between activity, and there was no differences in the composition and density of meio-epifauna between habitats within activity. Similarity Percentage (SIMPER) analysis was next performed in order to determine which groups were responsible for the differences. These analyses were performed using PRIMER (Plymouth Routines in Multivariate Ecological Research) v. 7.0 programme.

Review of meiofauna and macrofauna in wood substrates. A review and descriptive analysis were made for the wood-associated fauna in cold seeps and hydrothermal vents reducing environments. The aim was to provide an understanding of the connections between these two types of ecosystems. Information on composition, densities, locations and depths was gathered.

3.3 Results

3.3.1 Wood block description

After 10.5 months of deployment, the wood blocks at both the active and inactive sites were almost intact. Neither any wood degradation process nor bivalve drilling was found. No galleries or softening were observed, and the initial wood coloration remained (Fig. 3-1). On-board, when blocks were retrieved, macro-organisms associated to the wood (e.g., crabs, shrimps, mussels, limpets, Provanna gastropods, polychaete tubes, ophiuroids, chitons, anemones, among others), and some other associated to the mesh (e.g., yeti crabs and terebellids polychaetes), were observed. Patches of microbial mats were observed on different places of the blocks, and a possible carbonate encrustation was seen on Wood# 8 (observations from cruise data).

3.3.2 Meio-epifaunal composition

After 10.5 months of deployment, the wood blocks meio-epifauna was represented by a total of nine higher taxa, including permanent and temporary meio-epifauna (Fig. 3-2). A total of 9,951 individuals were recorded (Table 3-2). Individual wood blocks ranged from 2.9 (on an active site) to 26.3 (on an inactive site) individuals per 10 cm-2. Permanent meio-epifauna accounted for 85 % of the total number of organisms, including Copepoda, Nematoda, and Ostracoda, while temporary meio-epifauna, such as mollusks and polychaetes, represented the remaining 15 %. Crustacean nauplii were included in the counts as they were constant and numerous in all samples. Foraminiferans and other invertebrate groups were absent from the samples.

Of the major meio-epifaunal taxa, copepods were the most abundant group (75.1 %) (Fig. 3-2). Next in abundance were nematodes (9.8 %) and polychaetes (1.7 %). A single specimen each was found for Kinorhyncha and Chaetognatha. For the temporary forms, nauplii dominated in abundance (11.7 %), followed by bivalves (0.8 %) and juvenile gastropods (0.6 %). Temporary fauna occurred in all blocks, except for bivalves that were not present in Wood #1, situated at a mussel bed, active site.

Total mean density on wood was 13.6±7.0 ind.10 cm-2 (mean±1 SD). Grouping by seepage activity, the highest mean density was found in inactive sites (17.6±7.8 ind.10 cm-2) (Fig. 3-3), while in active sites it was 10.6±5.3 ind.10 cm-2. According to the habitat, near mussel beds (inactive) had the Chapter 3 57 highest number of associated individuals, with 26.3 ind.10 cm-2 (although no replica was available for this habitat owing to the lost block #3) (Fig. 3-4).

In all seven wood blocks the most prominent taxon was Copepoda (mostly Harpacticoida), with high relative abundances, from 48.8 % (Wood #1) to 94 % (Wood #5) of individuals, followed by Nematoda, from 0.3 % (Wood #6) to 26.5 % (Wood #8) (Table 3-2). According to the seepage activity, the highest mean density of copepods was found in inactive sites (12.4±7.1 ind.10 cm-2), while in active sites it was 8.5±5.4 ind.10 cm-2.

Active Inactive

100%

90%

80% Kinoryncha Chaetognatha 70% Nauplii 60% Ostracoda

50% Gastropoda Bivalvia 40% Polychaeta 30%

Nematoda Relative abundance Relative abundance (%) 20% Copepoda

10%

0% W#1-A-MB W#4-A-MB W#5-A-TW W#6-A-TW W#2-I-NMB W#7-I-RB W#8-I-RB

Figure 3-2: Relative abundance (% of total density) of different taxa of meio-epifauna found on each wood block. Abbreviations of each block are Wood number (W#), seepage activity (A=Active, I=Inactive), and habitat (MB=Mussel bed, NMB=Near mussel bed, RB=Rubble, TW=Tubeworms).

58 Meio-epifauna Costa Rica

100% Kinoryncha 90% Chaetognatha 80%

70% Nauplii

60% Ostracoda

50% Gastropoda

40% Bivalvia

30% Polychaeta

20% Nematoda Relative abundance Relative abundance (%) 10% Copepoda 0% Active Inactive

Figure 3-3: Relative abundance (% of number of total meio-epifaunal density) per taxa combining all wood blocks by seepage activity.

Active Inactive

100% Kinoryncha 90%

80% Chaetognatha

70% Nauplii 60% Ostracoda 50% Gastropoda 40% Bivalvia 30% Polychaeta 20%

Relative abundance Relative abundance (%) Nematoda 10% Copepoda 0% MB TW NMB RB

Figure 3-4: Relative abundance (% of total meio-epifaunal density) per taxa combining wood blocks for each habitat. MB=Mussel beds, NMB=Near mussel bed, RB=Rubble, TW=Tubeworms. Chapter 3 59

3.3.3 Community analysis

Cluster analysis and MDS of the seven wood block densities of meio-epifaunal taxa resulted in a wide separation of Wood #1 from the other blocks (Fig. 3-5 A, B). Wood #1, located in an active mussel bed was characterized by its low total number of taxa and low abundance (5 taxa, 2.93 ind.10 cm-2) rather than by a different composition of the main taxa (see Fig. 3-2). Also, Wood #1 comparatively had the highest number of polychaetes in the survey. Although the remaining samples had relatively high similarity in the cluster analysis (Fig. 3-5 A) and closely grouped in the MDS (Fig. 3-5 B), and were found to correspond to be the same assemblage according to the SIMPROF test (Fig. 3-5 A, only black lines in the dendrogram indicate groups that are established), blocks were segregated by seepage activity at 84 % similarity level in the cluster analysis (Fig. 3-5 A). At a greater level of similarity, the replicate blocks located in rubble bottoms (inactive) and in tubeworms were similar to each other, indicating similarity within habitats in each activity.

Owing to the outlier nature of Wood # 1, and the relatively high similarity among the remaining blocks, the two-way nested ANOSIM test showed no significant differences between blocks located in active vs. inactive sites (global R=0.5, significant level p=0.3) and habitats within seepage activity (global R=0.5, significant level p=0.1). Therefore, the null hypotheses of no significant differences in the composition and density of meio-epifauna between seepage activities, and between habitats within activity, could not be rejected. The SIMPER analysis showed an average dissimilarity of only 21.6 % between active and inactive blocks, with Nematoda being the main responsible for dissimilarities between both groups (contribution 27.4 %) (Table 3-3) being more abundant in woods located in inactive sites.

Table 3-2: Total abundance (standardized density, individuals in 10 cm-2 in parentheses) of meio-epifauna colonizing wood blocks for 10.5 months on Mound 12 - Costa Rica. Total percent (%) contribution of each group is also included. A=Active, I=Inactive, MB=Mussel bed, NMB=Near mussel bed, RB=Rubble, TW=Tubeworms. TA=Total abundance.

Wood Wood Wood Wood Wood Wood Wood #1 #4 #5 #6 #2 #7 #8 Seepage TA+(Ind.1 % A A A A I I I activity 0 cm-2) Total Habitat MB MB TW TW NMB RB RB 150 805 1501 1113 2155 760 985 7469 Copepoda 75.0 (1.4) (7.7) (14.3) (10.6) (20.6) (7.3) (9.4) (71.3) 38 10 25 4 250 233 418 978 Nematoda 9.8 (0.4) (0.1) (0.2) (0.0) (2.4) (2.2) (4.0) (9.3) 84 23 16 13 23 2 12 173 Polychaeta 1.7 (0.8) (0.2) (0.2) (0.1) (0.2) (0.0) (0.1) (1.6) 0 2 8 10 20 33 11 84 Bivalvia 0.8 (0.0) (0.0) (0.08) (0.1) (0.2) (0.3) (0.1) (0.8) 0 3 10 0 0 10 1 24 Ostracoda 0.2 (0.0) (0.0) (0.1) (0.0) (0.0) (0.1) (0.0) (0.2) 24 14 5 1 6 5 1 56 Gastropoda 0.6 (0.2) (0.1) (0.0) (0.0) (0.1) (0.0) (0.0) (0.5) 0 0 0 0 0 1 0 1 Kinorhyncha 0.01 (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.01) 60 Meio-epifauna Costa Rica

0 1 0 0 0 0 0 1 Chaetognatha 0.01 (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.0) (0.01) 11 335 31 190 304 150 144 1165 Nauplii 11.7 (0.1) (3.2) (0.3) (1.8) (2.9) (1.4) (1.4) (11.1) 307 1193 1596 1331 2758 1194 1572 9951 Total 100% (2.9) (11.4) (15.2) (12.7) (26.3) (11.4) (15.0) (95.0) N° Taxa 5 8 7 6 6 8 7

Table 3-3: Dissimilarity results calculated by SIMPER comparing between seepage activity. Taxa contributing most to the dissimilarity between groups resulting from the cluster analysis. Av- =average, Abund=abundance in the cluster group, Diss=dissmilatiry, SD=standard deviation, Contr=contribution, Cum=cummulative, A=Active, I=Inactive. Nematodes are the most important contributors to dissimilarity, being more abundant in woods located in inactive sites.

Group A Group I Taxa Av. Abund Av. Abund Av. Diss Diss./SD Contrib. % Cum. % Nematoda 0.6 1.3 5.9 4.1 27.4 27.4 Copepoda 1.6 1.8 3.1 1.1 14.2 41.6 Nauplii 0.9 1.2 2.9 1.3 13.4 55.0 Bivalvia 0.4 0.7 2.7 1.1 12.4 67.4 Ostracoda 0.2 0.3 2.3 1.2 10.9 78.3

A.

Group average Transform: Fourth root Resemblance: S17 Bray-Curtis similarity 60 Activity A I

70

y

t

i

r

a l

i 80

m

i S

90

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B B B B B

W W

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M M M

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Chapter 3 61

B.

Non-metric MDS Transform: Fourth root Resemblance: S17 Bray-Curtis similarity 2D Stress: 0,01 Similarity 70 Activity A I

W4-A-MBW7-I-RB W1-A-MB W5-A-TWW6-A-TWW2-I-NMBW8-I-RB

C.

Figure 3-5: Cluster (A) and MDS ordinations (B-C) of pairwise Bray-Curtis similarity coefficients of meio-epifaunal assemblages of wood blocks after 10.5 months of deployment. Data standardized to numbers of individuals per 10 cm2, and fourth root tranformed. (Red=Inactive sites, Blue=Active sites). The two groups produced by a threshold similarity of 70 % are shown. The group of blocks depicted by a continuous red line is homogeneous and significantly different from the other, from SIMPROF. (C) MDS zoom of the tightly grouped blocks, having the same pattern of active vs. inactive segregation found in the cluster analysis.

3.3.4 Wood associated meiofauna and macrofauna on reducing deep-sea environments

Experimentation with and observation of organic substrates in reducing environments (cold seeps and hydrothermal vents) are not new. Worldwide efforts have been made in order to standardize experimental substrates used to obtain comparative data. Deep-water information related to experimentally immersed wood substrates are supported by the results of Turner (1977), Maddocks and Steineck (1987), Lorion et al. (2009), Bernardino et al. (2010), Gaudron et al. (2010), Bienhold et al. (2013), Cunha et al. (2013), Grupe (2014), among others. Some of those studies evaluate specific colonizer groups, such as mollusks; others analyze the chemical characteristics of the wood. However, most of them have in common that they were developed to evaluate macrofauna. Appendix 1 shows a review of the information of two main taxonomic groups of the meiofauna (nematodes and copepods), recording their abundances or densities when available, locations, and depht. Also, the macrofauna (as defined by Grupe, 2014) is included along with our own observations in Mound 12 of Costa Rica. In total meiofauna, for both the Pacific and the Atlantic waters, about 70 taxa are 62 Meio-epifauna Costa Rica known, many of which remain undescribed to the species level, and which will require complex and extensive taxonomic work.

3.4 Discussion

Wood characteristics. An important factor when working with organic substrates is their durability at the bottom before being totally degraded. Bernardino et al. (2012) found that the more labile organic material, such as kelp, is scavenged by invertebrates and decomposed by microbes at much higher rates than wood falls of similar mass. The occurrence of a sulphophilic phase (i.e., organic material breakdown over large time periods) leading to the development of a reducing habitat with measureable concentrations of hydrogen sulfide, providing suitable habitat for “sulphur-loving” species (Simth & Baco, 2003; Levin et al., 2016). The organic-fall succession can last for at least 5 to 6.8 years for wood and whale, but it is much briefer for more labile plants (e.g., 0.5 years for kelp) (Bernardino et al., 2012; Cunha et al., 2013). Also, Cunha et al. (2013) in the Gulf of Cadiz, found that substrate consumption was faster in alfalfa than in wood, and in shallower than in deeper sites, but in most cases a large amount of the initially deployed volume of substrate remained after 1 to 2 years. As Cunha et al. (2013) points out, lability of the vegetable substrate is one of the reasons that explain the state of degradation, and the duration and development of the ecological succession.

In the case of wood, factors such as tree species, hardness, and texture, could affect the presence or absence of a particular faunal group. Experimental studies of macrofaunal succession have confirmed the relatively slow decomposition of wood compared to other vegetable remains (Bernardino et al., 2010; Bienhold et al., 2013; Cunha et al., 2013; Laurent et al., 2013). In our case, the selected wood Douglas fir is catalogued as a very hard and strong wood, with medium to coarse texture. Its heartwood is rated to be moderately durable regarding decay (http://www.wood- database.com/lumber-identification/softwoods/douglas-fir/). In the short term, this wood has shown very good results in other colonization experiments carried out in Atlantic Ocean waters (as analyzed below in the document). Douglas fir is not a native tree of Central America. It is distributed from British Columbia (Canada) to Mexico (Grandtner, 2005), however this tree has been used in reforestation in other countries, mainly in Europe, and is one of the most important timber trees, largely used in carpentry. Although it not found naturally in Costa Rica, it would not be unusual to find this or other types of wood not identified, as observed during the research cruise on Costa Rican bottoms.

Wood chemistry. Chemical variations are expected to occur in the wood substrates, although they have not been measured. Once the substrates reach the ocean, complex chemical processes on their surface begin. Studies have found a relationship between some colonizers and wood chemical conditions. Degradation processes in organic falls can lead to reducing conditions and high sulfide concentrations, attracting chemoautotrophic bacteria, both free-living and as symbionts of chemosynthetic fauna (Bienhold et al., 2013). Sulfide is a principal product of the bacterial degradation of wood in marine environments (Distel et al., 2000). “This can be produced from wood from synergetic microbial activities involving cellulolytic bacteria and sulfate-reducing bacteria with or without the mediation of wood borers, at shallow to great depth” (Laurent et al., 2013). These measurements are not common in situ deep water, primarily because of the logistics involved. Direct Chapter 3 63 measurement of some chemical variables on woods in shallow waters has found that sulfide varied substantially throughout the experiment duration (Laurent et al., 2013). Laurent et al. (2013) compared Rhizophora mangle and Cocos nucifera and found both types of wood exhibit a rapid (four and six days respectively) decrease in the electrode potential, denoting the establishment of sulfidic conditions on their surface; these authors also found that sulfide remained undetectable in the water surrounding the immersed substrates. The latter would imply that it is necessary to measure directly on the wood, and that it is not useful to do so in the surrounding water.

In another example using Pine wood, laboratory experiments simulating deep water conditions of the Atlantic Ocean (500-1000 m) established that the highest sulfide levels on the wood surface have been measured between days 59 and 64 (5–10 µM). It also found white spots on the surface around day 60, the presence of microbes typical of sulfidic habitats, and progressive coverage of the wood surface with digested materials expelled from wood-borer burrows after day 130 (Yücel et al., 2013).

Possibly due to the type of wood used (Douglas fir), the length of time the wood substrate was left on the bottom (10.5 months), and the conditions of this sector of the Pacific, the processes of degradation and chemical alteration that have been established in other experiments were not observed. In laboratory experiments with Mediterranean seawater, cellulolytic activities were reported in fir wood, mainly in the first months of experimentation; also, in parcels implanted at 1243 m and 1675 m depth for 4.5 yr and 1.8 yr, respectively (Palacios et al., 2006). In the latter study, cellulolytic activities varied significantly with the wood source and its original location. Although no measurements to detect the degradation of wood were carried out in our study, we observed that fir wood blocks were almost completely intact (Fig. 3-1), and that degradation processes could be slower due Eastern Pacific environmental conditions (i.e., low oxygen in the overlying water), and the natural wood resistance. Thus, the presence in some blocks of bacterial mats or attached seep macrofaunal elements may be due more to the fact that samples were placed in a natural seep environment, and not to the fact that the wood itself had contributed to the reducing conditions through decay.

Wood borers role. The leading role of wood borers in the utilization of woody substrates in the deep sea is already known (Laurent et al., 2013). In deep water biogenic substrates, initial colonizers, like bacteria and wood-boring bivalves (subfamily Xylophagainae), may provide food sources for the following successional fauna, while at the same time degrading the substrate (Schwabe et al., 2015). Bacteria, fungi, and Xylophagainae provide the link in the specialized food chain by converting wood into animal tissue, fecal pellets and macerated, decomposed debris, which can be utilized for other invertebrates (Turner 1973, 1977; Maddocks & Steineck, 1987). These decomposers increase the area available for colonization by other macrofaunal species (Gaudron et al., 2010). At a small scale, such three-dimensional habitats also favor a rich assemblage of other organisms of meio- epifauna. However, these processes were not evident in our samples.

Compared with Gaudron et al. (2010) and Bienhold et al. (2013) studies, carried out in deep water chemosymbiotic ecosystems (cold seeps and hydrothermal vents) of the Atlantic Ocean, during 10- 12 months of deployment and using the same wood (Douglas fir), both studies found wood had been degrading by the activity of macrofaunal wood-boring bivalves (i.e., Xylophaga dorsalis, X. atlantica and Xyloredo ingolfia). Wood-boring bivalves are well adapted for this short-term lifestyle, 64 Meio-epifauna Costa Rica and high settlement was observed after only two weeks in wood deployed in the Eastern Mediterranean (Gaudron et al., 2010; Cunha et al., 2013). Cunha et al. (2013) found that after 2 years, approximately 60 % of the substrate was consumed by the action of the woodboring bivalve X. dorsalis in one area of experimentation, while in another they found that the low colonization by xylophagainin bivalves and lysianassid amphipods may be explained by the low level of degradation of the organic substrate in deeper mud volcanoes (10-30 % of the material was consumed).

It is also interesting to mention the spatial differences in the colonization pattern observed across the substrate. Bienhold et al., (2013) found “the state of degradation and colonization differed between wood logs, but also between different positions on one log. Those sides of the wood logs lying in the sediments appeared to be less colonized and less degraded by wood-boring animals than those exposed to the bottom waters. The degradation of the wood was mainly due to the activity of X. dorsalis”.

Faunal relationship with wood. Distel et al. (2000) proposed several reasons why decomposing wood supports chemosynthesis-based invertebrate communities while other substrates do not. Among these are: wood has a high reduced-carbon content, resists consumption by detritivores, and undergoes slow and sustained decay. The latter could be necessary to support these species, which may require several years to reproduce. Organic falls (by wood and other plant remains) are fundamental to the nutritional ecology of the deep sea by the direct or indirect mobilization of organic carbon, the increased flux of reduced chemicals, and their potential role as nitrate sinks (Cunha et al., 2013).

Most of the deep seafloor receives a very low supply of energy and nutrients, leading to extremely oligotrophic conditions in extensive areas of the ocean. Substrates such as sunken wood, whale carcasses, kelp and other food fall, offer locally and temporally restricted inputs of organic material to the deep sea, which are quickly localized and exploited by opportunistic fauna, and which rapidly develop into hotspots of diversity (Bienhold et al., 2013). Although most of the community analyses on reducing environments has focused on macrofaunal communities (see Introduction for references), it is known that not only do large numbers of invertebrates occupy these micro- topographically complex habitats, but also highly diverse assemblages would seem to have evolved with specific adaptations to exploit this extreme environment (Hicks, 1988). From the perspective of the macrofauna, in this study, different components that have chemosymbiotic associations were observed attached to or on the wood blocks (Fig. 3-1). However, the substrate was not entirely colonized, and thus the results of the current study represent primary stages of succession.

Studies on shallow water wood colonization and fauna associated and chemical conditions, found that the best represented groups were oligohymenophoran, heterotrich ciliates, annelids, and arthropods (Laurent et al., 2013). Laurent et al. (2013) found also among first colonizers, sulfide- tolerant species, that dominated over several weeks when the sulfide content was maximum. When sulfide decreased, these were followed by less tolerant opportunistic species. Nematodes were occasionally present on the surface of the wood, but did not exhibit a marked preference for sulfide- rich stages on wood (Laurent et al., 2013). Other studies with two different types of submerged wood (natural collected wood at 560-580 m depth) found completely different colonization patterns possibly related to differences in chemical composition or to time elapsed since sinking (Pailleret et Chapter 3 65 al., 2007). In our study (see Chapter 4) using 20 biogenic and non-biogenic surfaces as woods pieces, carbonates rocks, polychaetes tubes and clam shells, it was found that in all habitats and substrates surveyed on Mound 12 (after 10.5 months of colonization), the most prominent taxon within meio- epifauna was Copepoda, with 73.3 % of total abundance. The latter could be related to a first colonization phase dominated by these organisms.

Meio-epifauna composition and assemblage at Mound 12. Like other experiments carried out using different substrates, low diversity of higher taxa was observed (only nine groups) in the present study. Bright et al. (2010), in the Gulf of Mexico between depths of 1400 to 2800 m, documented epizooic communities composed of a limited number of higher taxa (only five), including Nematoda, Copepoda, Ostracoda, Halacarida, and Kinorhyncha. Meanwhile, Gaudron et al. (2010) working with meio and macrofauna, of the typical meiofaunal contents, it only listed Copepoda with a low density related to the other groups (12.5 and 6.3 individuals per dm3) within the devices (using wood and alfalfa substrates). About other listed taxonomic groups in such study it is not clear if they correspond by their size to meiofauna or to macrofauna (e.g. Mollusca, Polychaeta, Nemertea, Sipuncula, etc.).

Surprisingly, copepods were the most abundant taxon on our wood blocks. These results are significant if one considers that in most of the cold seeps nematodes are identified as dominant in meiofaunal communities. Similar results were found for authigenic carbonate rocks deployed simultaneously and for the same duration at Mound 12, as described in chapter 2. We could thus conclude that during the early phase of colonization of hard wood, while there is little decomposition, colonization proceeds as it would in any hard substrate, as products from wood decay can influence particular bacterial or faunal groups may not yet have occurred within the 10.5 months of immersion, in this type of wood and in this particular area.

From these observations and results, some questions remain to be answered: Are copepods dominant in the early stages of succession of any hard substrates in the east Pacific deep sea? Do copepods follow a pattern of wood colonization consistent with the chemical evolution of the wood, or is it more related to the appearance of competitors and predators as time progresses? Are they dominating when sulfide levels are the lowest? Are the characteristics of the basin (Atlantic vs. Pacific Ocean) related to their dominance?

Although in this work copepods were not identified to a lower taxonomic level, there are several known factors that explain, in the case of harpacticoid copepods for example, their success in hard substrates. It has long been known that harpacticoid copepods could live on several hard substrates, as well as dwelling on polychaete tubes, seagrass blades, macro algae, and mudballs (see references in Thistle & Eckman, 1988). In the upper food web these copepods serve as food for many invertebrates and juvenile fishes. In the lower portion of the web, the suggestion is that these copepods feed to a large extent on the microbial flora and are therefore crucial in the recycling of benthic energy (Decho, 1983). Copepods feed on a broad organic spectrum of organic detritic particles (i.e., microalgae, protozoans, bacteria) (Rieper, 1982; Buffan-Dubau et al., 1996), factors that are known to affect their distribution (Rieper, 1982). In this sense, benthic copepods could represent a significant link in the cold-seep food web in Costa Rican deep-sea areas. 66 Meio-epifauna Costa Rica

Note that the pattern found in our study is not consistent with other studies conducted for the same time length of colonization. Zeppilli et al. (2015) in a hydrothermal vent field of the Atlantic Ocean observed that nematodes colonized both organic (wood and bone) and inorganic substrates in high abundance and diversity after just nine months. The highest value found in wood was 242,006.1 ind. m-2, when the substrate was located in an external position in relation to the emission of fluids. Although the pattern of colonization between nematodes and copepods is not clear, at least by comparing subpatterns with information on shallow data, some authors propose that nematodes do not seem to be attracted by sulfide-rich habitats on wood, whereas they dominate the mangrove sediment meiofauna (Kathiresen & Bingham, 2001 in Laurent et al., 2013).

To try to solve some of these questions, information from hydrothermal vent and cold seeps in different kinds of susbtrates could be contrasted because these showed significant copepod abundance. In the Juan de Fuca Ridge (Pacific Ocean), Tsurumi et al. (2003) found densities between 0.5 copepod cm-2 (=5 ind.10 cm-2) on vestimentiferan tube surfaces to over 8 cm-2 (=80 ind.10 cm-2) on chimney surfaces. The second value is considered high in relation to our data: 1.4 ind.10 cm-2 on Wood#1 (active area) and 20.6 ind.10 cm-2 Wood#2 (inactive area). Bright et al. (2010), studying meiobenthos associated with tubeworm and mussel aggregations (cold seep - Gulf of Mexico), recorded more organisms per unit area: 1 to 81 individuals per 10 cm-2 sample area, in contrast to our data: 2.9 ind.10 cm-2 on Wood#1 - active area; and 26.3 ind.cm-2 Wood#2 - inactive area. Copepods, second in densities after Nematoda, also had higher densities than our study, ranging between 0.2 and 73 ind.10cm-2 (Bright et al., 2010). Otherwise, experiments involving different substrates (i.e. wood, alfalfa, carbonates) found copepods only in organic substrates: after 1 yr, 6.3 ind/dm3 were reported for cold seeps, both on wood and alfalfa, and after 2 weeks 12.5 ind/dm3 were reported for cold seeps on woods (Gaudron et al., 2010). However, in this case the data obtained are not comparable because the measurement units used were different.

In relation to the time elapsed in colonization process in hydrothermal vents, Copley et al. (2007) studying mussel beds, found nematodes were the numerically dominant meiofaunal taxon at the majority of sites (in 5 of the 7 studied, ranged in abundance between 57-74 % of all fauna), but copepods were the most abundant at two stations (ranged between 55 and 75 %), which were representing the youngest mussel beds at the time of sampling (4 and 5 year age). These results may suggest that community composition changes with the residence time of the substrate, and also a possible negative relationship between substrate age and copepod abundance.

Differences between habitats and seepage activity in our study were not significant; these results may be reflecting the same early state of succession among all wood blocks, because all remained submerged for the same amount of time. However, the lack of significance in ANOSIM could be due to low numbers of samples used (Clarke & Gorley, 2015). Although no significant differences between seepage activity, and habitats within the activity were found, there is segregation for most of the samples between active and inactive sites. This is probably a result of a low sample size. However, this allows detecting a pattern that can be corroborated in future studies.

On the other hand, copepods are known to be restricted to the oxygenated regions in sediments. In that environment, they tend to be found on or just beneath the surface of muds (Wells, 1988). This fact is reaffirmed by Wetzel et al. (2001) who explained that vertically in muddy sediments, total Chapter 3 67 meiofaunal abundance typically declines with increasing sediment depth through the upper 4 - 6 cm. But on a fine scale, however, harpacticoids are the most abundant meiofaunal taxon in the upper 4 mm, while many nematodes species have deeper abundance maxima. In this sense, we found the highest mean density of copepods occurred in the inactive areas, which could demonstrate their preference for better oxygenated places; for cold-seep sediments are considered extreme environments where the associated fauna is exposed to high levels of toxic hydrogen sulfide, high concentrations of methane and low oxygen concentrations (Sibuet & Olu, 1998; Levin, 2005; Cunha et al., 2013). Powell et al. (1986) at brine seeps in the Gulf of Mexico found that outside the influence of the seep, meiofaunal copepods predominate. They determined that nematodes exploit sulfide systems considerably better than copepods (as an increase in sulfide concentration will increase the relative importance of nematodes), which disagree with discussed above for shallow-water nematodes associated with mangroves (see Laurent et al., 2013). Hence, much of the significance of the nematode/copepod ratio can be reinterpreted based on the relative importance of sulfide-tolerant organisms in the community. Therefore, future studies must take into account the chemical variations presented in each environment and over the substrate, as these may be what are actually structuring the community.

In comparing Atlantic vs. Pacific Ocean basins, Zekely et al. (2006) worked in vents, studying mytilid mussel aggregations in the East Pacific Rise-EPR and the Mid-Atlantic Ridge-MAR, where they found communities composed of nematodes, copepods, ostracods and mites. Communities at both of these areas showed low species richness (24 species at EPR, 15 species at MAR), low diversity, and low abundance (32±4 ind.10cm-2 at EPR, 43±5 ind.10 cm-2 at MAR). Although copepods were abundant, composition was remarkably different between the two areas. At MAR, nematodes dominated (63±19 %) followed by copepods (35±4 %); at EPR, copepods (85±2 %) were more abundant than nematodes (6±2 %). In this case, the authors hypothesize that the abundance of meiobenthos at hydrothermal vents is strongly influenced by the macrobenthos.

The total abundance of nauplii was remarkable in this study (Table 3-2). Unfortunately, nauplii have been excluded from the majority of cold seeps and hydrothermal vent ecological analyses because they could not be assigned to aspecific higher crustacean taxon (e.g., Bright et al., 2010; Degen et al., 2012). To Atlantic Ocean, Van Gaever et al. (2009), examined the spatial variation and distribution patterns of the meiobenthos in different seepage-related habitats in two regions off Norway finding that after nematodes, adult copepods and their naupliar larvae always constituted the second most abundant group, showed nauplii densities between 0.2 ind.10cm-2 (Storegga site, Siboglinidae area) to 169.7 ind.10cm-2 (Nyegga site, Siboglinidae area). While on hard substrates, Plum et al. (2017) recorded densities for all copepod species collected from samples of three different substrates deployed at five stations along a gradient of hydrothermal activity, showed densities between 0.006 ind.10cm-2 (in an inactive site – slate substrate) to 52.364 ind.10cm-2 (low emission – wood substrate); they didn't find nauplii in a high emission site (wood substrate), a external emission site (both slate and bone substrates), and an inactive site (wood substrate). Also, they analyzed that the individual densities of adults, copepodites, and nauplii revealed similar patterns although the relative abundance of the individual developmental stages showed high variability across all sites. According these same authors the discovery of high abundance of juvenile copepod stages contradict previous assumptions that copepod larval stages only inhabit micro- habitats 68 Meio-epifauna Costa Rica outside the hydrothermal influence, and their experiment confirms that copepod communities can be well established in a variety of microhabitats and are capable to spend their whole life cycle under extreme conditions. Conclusions that could also be generalized for what we found in Costa Rican study on cold seeps.

What do we know about wood meio-epifauna and macrofauna at Costa Rica and other places? The relationships of macrofauna and meiofauna should be explored primarily from a food web perspective, as these groups of organisms are closely linked throphically. In our study area, Grupe (2014) found that macrofauna colonized experimental wood and carbonate substrates at higher densities in sites of seep fluid flow than in inactive sites (but at active seeps, carbonates contained significantly higher densities of colonizing macrofauna than did wood). This is the opposite of what we found for meio-epifauna. Macrofaunal gastropods were the most abundant group of colonizers associated with wood substrates in active and inactive sites. Different species had higher proportional abundances on different substrate-activity combinations. Provanna laevis was the most dominant species on carbonates (~55 % of all gastropods on Active, ~52 % on Inactive), but only made up ~20-30 % of the gastropods on wood and biogenic substrates. In contrast, Lepetodrilus guaymensis was proportionally more abundant on wood (~20 %) than other substrates. Taxa occurring in higher densities on wood on inactive substrates included ophiuroids, nemerteans, and cnidarians. According to Grupe (2014) seep activity significantly affected macrofaunal community colonizer composition, regardless of substrate type. The species contributing to dissimilarity between colonizers of active and inactive substrates were more abundant in active ones, mainly gastropods. To have a complete view of all the associated communities (mega, macro and meiofauna) inhabiting wood substrates, establishing trophic relationships and matter flow in this type of environment is an important objective.

Appendix 1. Nematodes and copepods found on wood substrates and other chemosynthesis-based habitats (vents and seeps). Also macrofauna recorded by Grupe (2014) found in woods at Mound 12. LSHV=Lucky Strike hydrothermal vent field. 1.Cuvelier et al. (2014)=Densities (ind/m2). 2.Schwabe et al. (2015)=Total number of individuals. 3.Zeppilli et al. (2015)=Relative abundance (%). 4.Gaudron et al. (2010)=Densities per dm3. 5Plum et al. (2017)=Densities (ind.10 cm-2), the depth range is from the entire experiment. * Seep endemic gastropod.

Main Taxa Genus/ Abundance Ecosystem/ Depth Family Source Meiofauna species /density habitat (m) Nematoda Class Aenophora Undeterminated sp. 253.1 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Class Enoplea Order Enoplida Anticomidae Anticoma sp. 13 North West Pacific ~5300 2Schwabe et al. (2015) Oncholaimidae Oncholaimus sp. 2 North West Pacific ~5300 2Schwabe et al. (2015) Oncholaimidae Oncholaimus sp. 1 102.2 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Ironidae Syringolaimus sp.1 2.2 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015)

Class Chromadorea Order Rhabditia Anguinidae Anguinidae gen. sp1 23 North West Pacific ~5300 2Schwabe et al. (2015) Order Chromadorida Cyatholaimidae Paracanthonchus 261.2 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Cyatholaimidae Paracanthonchus sp.2 2.2 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Chromadoridae Acantholaimus aff. quintus 1 North West Pacific ~5300 2Schwabe et al. 2015) Chromadoridae Endeolophos sp. 1 North West Pacific ~5300 2Schwabe et al. (2015) Chromadoridae Chromadoridae gen. sp. 2 1 North West Pacific ~5300 2Schwabe et al. (2015) Chromadoridae Chromadoridae gen. sp. 3 1 North West Pacific ~5300 2Schwabe et al. (2015) Order Desmodorida Draconematidae Cephalochaetosoma 17461.3 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Draconematidae Cephalochaetosoma sp.1 22.2 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Draconematidae Dinetia 1983.7 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Epsilonematidae Epsilonema 98 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Epsilonematidae Epsilonema sp.1 8.9 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Microlaimidae Microlaimus 1453.1 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Microlaimidae Microlaimus sp. 1 North West Pacific ~5200 2Schwabe et al. (2015) Microlaimidae Microlaimus sp. 1 21.5 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Microlaimidae Aponema sp.1 18.5 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Microlaimidae Calomicrolaimus sp.1 10.4 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Order Monhysterida Monhysteridae Halomonhystera 1975.5 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. 2014 Monhysteridae Halomonhystera sp.1 115.6 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015)

70 Meio-epifauna Costa Rica

Monhysteridae Halomonhystera sp.2 40 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Thalassomonhystera Monhysteridae 49.6 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) vandoverae Xyalidae Cobbia 8.2 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Xyalidae Cobbia sp.1 2.2 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Xyalidae Theristus sp. 1 4.4 LSHV Mid-Atlantic Ridge 1700 3Zeppilli et al. (2015) Nematoda 7 ind. North West Pacific ~5300 2Schwabe et al. (2015) Copepoda 12.5 (After Nile Deep sea Fan Cold seep. Copepoda spp. 1693 4Gaudron et al. (2010) 2 weeks) Eastern Mediterranean 6.3 (After 1 Haakon Mosby Mud volcano. Copepoda spp. 1257 4Gaudron et al. (2010) year) Atlantic Ocean Calanoida Calanoida sp. 1 0.007 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Cyclopoida Cyclopoida sp. 0.007 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Cyclopinidae Heptnerita confusa 0.068 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Undetermined spp. 138.8 LSHV Mid-Atlantic Ridge 1Cuvelier et al. (2014) Harpacticoida Ameiridae Ameira longipes 17.08 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Ameiropsis mixta 0.663 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Nitroca sp. 8.6 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Haifamera sp. 26.606 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Nitocrella sp. 166.165 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Ameiridaesp.4 10.887 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Undetermined spp. 653.1 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. 2014 Ancorabolidae Lobopleura expansa 0.175 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Lobopleura cf. expansa 220.4 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Cletopsyllidae Retrocalcar sp. 0.004 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Ectinosomatidae Ectinosomatidae sp.2 2.187 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Ectinosomatidae sp.5 0.004 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Undetermined spp. 416.3 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Laophontidae Archesola typhlops 0.32 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Bathylaophonte azorica 4.089 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Miraciidae Miraciidae sp. 1 0.184 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Miraciidae sp. 2 11.561 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Miraciidae sp. 3 0.108 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Amphiascus spp. 1828.6 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Chapter 3 71

Pseudotachidiidae Xylora bathyalis 11.527 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Xylora cf. bathyalis 318.3 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Donsiella cf. bathyalis 1657.2 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Tegastidae Smacigastes micheli 15.187 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) 10138.8 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Tisbidae Tisbe sp. 16.204 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Tisbe spp. 122.5 LSHV Mid-Atlantic Ridge 1700 1Cuvelier et al. (2014) Poecilostomatoida Erebonasteridae Ambilimbus arcuscelestis 0.003 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Ambilimbus sp. nov. 0.004 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Kelleriidae Kelleriidae n. gen. n. sp. 0.333 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Siphonostomatoida Siphonostomatoida Siphonostomatoidae sp.1 0.004 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) e Siphonostomatoidae sp.2 0.004 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Dirivultidae Aphotopontonius atlanteus 1.408 LSHV Mid-Atlantic Ridge 1695-1741 5Plum et al. (2017) Macrofauna Mound 12 - Woods Polychaeta Ampharetidae A Mound 12- Costa Rica ~1000 Grupe (2014) Cirratulidae A Mound 12- Costa Rica ~1000 Grupe (2014) Dorvilleidae A-I Mound 12- Costa Rica ~1000 Grupe (2014) (other) Euphrosinidae A Mound 12- Costa Rica ~1000 Grupe (2014) Flabelligeridae A-I Mound 12- Costa Rica ~1000 Grupe (2014) Hesionidae (other) A-I Mound 12- Costa Rica ~1000 Grupe (2014) Lacydonidae A-I Mound 12- Costa Rica ~1000 Grupe (2014) Nereidae A Mound 12- Costa Rica ~1000 Grupe (2014) Paraoinidae A-I Mound 12- Costa Rica ~1000 Grupe (2014) Polynoidae A Mound 12- Costa Rica ~1000 Grupe (2014) Spionidae I Mound 12- Costa Rica ~1000 Grupe (2014) Terebellidae A-I Mound 12- Costa Rica ~1000 Grupe (2014) Trichobranchidae A-I Mound 12- Costa Rica ~1000 Grupe (2014) Mollusca Mound 12- Costa Rica ~1000 Grupe (2014) Bivalvia Subclass Solemyidae Acharax sp. A-I Mound 12- Costa Rica ~1000 Grupe (2014) Protobranchia Nuculanidae Nuculanidae A Mound 12- Costa Rica ~1000 Grupe (2014) 72 Meio-epifauna Costa Rica

Yoldiidae Yoldiella sp. I Mound 12- Costa Rica ~1000 Grupe (2014) Subclass Mytilidae Bathymodiolus spp. A Mound 12- Costa Rica ~1000 Grupe (2014) Pteriomorphia Gastropoda Subclass Cocculiniformia Cocculiniformia A Mound 12- Costa Rica ~1000 Grupe (2014) Cocculiniformia Subclass Cataegidae Cataegis myronfeinbergi A-I Mound 12- Costa Rica ~1000 Grupe (2014) Vetigastropoda Lepetodrilidae *Lepetodrilus guaymensis A-I Mound 12- Costa Rica ~1000 Grupe (2014) Subclass Neolepetopsidae *Neolepetopsis sp. A-I Mound 12- Costa Rica ~1000 Grupe (2014) Patellogastropoda 1. *Paralepetopsis sp. A-I Mound 12- Costa Rica ~1000 Grupe (2014) Subclass Pyropeltidae *Pyropelta corymba A-I Mound 12- Costa Rica ~1000 Grupe (2014) Vetigastropoda *Pyropelta musaica A-I Mound 12- Costa Rica ~1000 Grupe (2014) *Pyropelta wakefieldi A-I Mound 12- Costa Rica ~1000 Grupe (2014) Juvenile Pyropelta spp. I Mound 12- Costa Rica ~1000 Grupe (2014) Subclass Provannidae *Provanna laevis A-I Mound 12- Costa Rica ~1000 Grupe (2014) Caenogastropoda Turridae Turridae A Mound 12- Costa Rica ~1000 Grupe (2014) Monoplacophora Monoplacophora Monoplacophora A Mound 12- Costa Rica ~1000 Grupe (2014) Polyplacophora Polyplacophora Polyplacophora A Mound 12- Costa Rica ~1000 Grupe (2014) Crustacea Amphipoda Amphipoda A Mound 12- Costa Rica ~1000 Grupe (2014) Caridea Caridea A-I Mound 12- Costa Rica ~1000 Grupe (2014) Galatheidae Galatheidae A Mound 12- Costa Rica ~1000 Grupe (2014) Kiva puravida A-I Mound 12- Costa Rica ~1000 Grupe (2014) Echinodermata Ophiuroid A-I Mound 12- Costa Rica ~1000 Grupe (2014) Nemertea Nemertea A Mound 12- Costa Rica ~1000 Grupe (2014) Cnidaria Anemone I Mound 12- Costa Rica ~1000 Grupe (2014)

4. Meio-epifaunal copepods (Crustacea) assemblages in experimental susbtrates at Mound 12 cold seep

Abstract

Copepods inhabiting deep-sea areas have been the object of recent and intensive investigations. Knowledge has accumulated from in situ colonization experiments conducted over recent decades at or near reduced ecosystems in the Atlantic and Pacific Oceans. The latter has resulted in solid characterization of the hydrothermal vent community; however, the understanding of copepod presence in cold seep areas is still limited. The Pacific continental margin offshore Central America shows a large number of cold seeps associated with faults, slump scars of submarine landslides, and mounds. At this convergent margin of Costa Rica, one of the best documented geomorphologies is Mound 12 (8° 55.77842’ N, 84° 18.73083’ W) located to the southeast of Nicoya Peninsula. To contribute to the understanding of copepod colonization processes in an area characterized by spatial heterogeneity, colonization experiments were conducted in active and inactive cold-seep sites of Mound 12. Experiments were deployed at ~1000 m depth from R/V Atlantis cruise AT15-44 (2009) and recovered after 10.5 months in 2010 during cruise AT15-59. Deployed substrates were composed of 21 biogenic and non-biogenic experimental units as wood blocks, carbonate rocks, siboglinid polychaete tubes and clam shells. A total of 24,467 individuals belongin to five orders and 15 families were identified. The order Harpacticoida was the best-represented regarding families (7) and densities (87.5 %). Miraciidae (38.6 %), Ectinosomatidae (34.2 %), Tegastidae (9.4 %) and Ameiridae (4.9 %), accounted for approximately 87.1 % of all copepods density, while Dirivultidae (Order Siphonostomatoida) added 7.3 %. The highest densities of copepods (16.4±9.9 (1 SD) ind.10cm-2, n=9) occurs in inactive areas, regardless of the type of experimental substrate. Multivariate analyses showed only one assemblage. At the family level analyzed, the assemblage responds to seepage activity. Also, a turnover in family dominance between Miraciidae and Ectinosomatidae, according to seepage activity, was observed. However, BEST analysis showed that families that best explain the assemblage are Aegisthidae, Ectinosomatidae, Eucalanidae, Miraciidae, and Tegastidae. It was also established that the type of substrate, especially wood and rock also conditions community structure.

Key words: Experiments, Authigenic carbonates, Wood blocks, Meio-epifauna, Pacific Ocean, Costa Rica, Copepoda, Colonization

74 Meio-epifauna Costa Rica

4.1 Introduction

One of the concerns that arise when trying to understand the diversity of organisms associated with extreme deep-sea environments, is how they develop and what are the main factors that are involved in their colonization process, whether on hard surfaces or in the sediments. Deep-water bottoms support an organic and inorganic substrate diversity (i.e., authigenic carbonates, sunken woods, animal bones, corals, mollusks shells and polychaete tubes, among others) that could be favorable for the development of small-sized organisms, which in turn support larger sized communities, following an elaborate trophic pattern.

Colonization processes on surfaces begin as soon as they are submerged in the ocean, being immediately subjected to an initial fouling process (Rittschof, 2000). For this process of colonization, three levels of organization have been recognized: a) Molecular fouling, b) Microfouling, and c) Macrofouling. That is, “it begins then with the accretion of organic and/or inorganic molecules from solution onto submerged surfaces. Subsequently follow the colonization of surfaces by micro-organisms, followed by the secretion of polymers that anchor and often embed the micro-organisms and other particles. And it ends up with the colonization of surfaces by macroscopic fauna and flora, usually by means of micro- or meiosized propagules” (Rittschof, 2000; Fonsêca-Genevois et al., 2006). However, these are not always linear; several studies have found that microfouling is not a prerequisite for macrofouling (Fonsêca-Genevois et al., 2006). Therefore, in colonization processes and especially in deep-sea environments, still many questions need to be answered.

Meiofaunal communities are one of the most significant components of diversity and biomass in all microhabitats, and it has been denoted their essential role in linking bacterial and macrofaunal food webs (Gollner, 2009). This function could be highly significant because bacteria are the basis of the food-web in reducing environments, as free-living organisms or as a faunal symbiont (Vrijenhoek, 2010). Meiofauna live on much smaller spatial and temporal scales, and mechanisms maintaining meiofauna diversity may be different than those for the larger-sized fauna (Baguley et al., 2006).

On a larger scale, cold seeps have been considered among the most heterogeneous habitats of continental margins (Cordes et al., 2010). This heterogeneity is provided by hydrographic (depth, temperature, O2), geochemical (gas activity, fluid flow intensity and volume), geological (tectonic activity, mud activity, diverse geomorphology, authigenic carbonates, geologic features age), and biological (microbial communities, foundations species,), all very contrasting conditions (Cordes et al., 2010).

At cold seeps, a variety of geologically diverse, reducing habitats can be distinguished by the presence of microbial mats, macro/megafaunal communities (Bright et al., 2010) and authigenic carbonates. It is also possible to observe other types of habitat, such as sunken woods or plant material that reaches these depths. Cold seeps are typically characterized by large megafaunal aggregations, such as bathymodiolid mussels and siboglinid tubeworms. These organisms can build large physical structures above the sediment surface. These hard substrates create habitat as foundation species for an associated macro- and meiofaunal community. Chapter 4 75

It is important to consider that habitat definition is scale-dependent, because relatively homogeneous variables measured at a given scale may appear heterogeneous at different observational scales (Levin, 1992; Cordes et al., 2010). Although cold seeps are known to occur on continental margins throughout the global ocean, variations in the composition of seep communities in different parts of the world are not well documented (Sibuet & Olu, 1998; Bowden et al., 2013). Much less if one considers the availability of information related to their spatial heterogeneity. Comparing meiobenthos between soft-bottom and epibenthic hard-substrate, less attention has been paid to the latter (Bright et al., 2010).

Meiofauna can be found both as mud-dwelling fauna, and also as group living epifaunally (or meio- epifauna) on surfaces ranging from large biogenic debris to seagrasses, macro-algae, larger coral fragments, sponge skeletons, manganese nodules, pebbles, etc. (Raes & Vanreusel, 2005). Within meio-epifaunal components, information on copepod composition related to substrates in the eastern Pacific cold-seeps is not available yet. This group can be considered as the insects of the seas regarding their diversity and abundance (Huys & Boxshall, 1991 in Shimanag et al., 2005). In general, they play a significant role in marine life (Heptner & Ivanenko, 2002), being usually the second dominant higher meiofaunal taxon (Giere, 2009; Plum et al., 2017) after the numerically dominant nematode group, although, this pattern is not always recurrent (see Chapters 2 and 3). They can be the most abundant and diverse meiofaunal taxon in some localities and conditions i.e., on hard substrates like basalts and sulfide chimneys present in hydrothermal vents (Plum et al., 2017). Their ecological importance has been documented, being among others, essential for nutrient recycling, abundant feeding source for macrofauna, and on the other hand, source for detritus feeders via their fecal pellets (Huys & Boxshall, 1991 in Shimanag et al., 2005; Gollner et al., 2010).

About 87 described species of Copepoda are known for reducing environments (i.e., hydrothermal vents and cold seeps), 79 from vents and eight from seeps (Heptner & Ivanenko, 2002; Ivanenko & Defaye, 2004a, b; Ivanenko et al., 2007; Gollner et al., 2008, 2010; Ivanenko et al., 2011; Ivanenko & Ferrari, 2013; Ivanenko & Martinez, 2016). They represent more than 15 % of the species documented from vents worldwide (Gollner et al., 2008, 2010). These species belong to orders Harpacticoida, Calanoida, Cyclopoida, Poecilostomatoida, Siphonostomatoida, and Misophrrioida; several authors agree that many more species await study and description (Heptner & Ivanenko, 2002; Gollner et al., 2010). By comparison, the number of species associated to cold seeps has deeper uncertainties, and colonization processes remain poorly understood. Ritt et al. (2010) argued “although there are some similarities between structures and regions, each newly discovered seep area may have its own signature regarding faunal structure, diversity and chemical conditions”. Such similarities could be reflected in finding similar mega and macrofaunal assemblages; however, in meio-epifauna, this is not clear yet.

To contribute to understanding colonization processes of meio-epifaunal copepods in places with physical and environmental heterogeneous characteristic in the Pacific cold seeps of Costa Rica, two questions are proposed:

• On a large scale, does the spatial heterogeneity given by such factors as habitat, seep's activity, and foundation species influence assemblages structure of meio-epifaunal copepods? 76 Meio-epifauna Costa Rica

• On a small scale, is meio-epifauna family copepod community structured by the diversity of colonization substrates available?

This work aims to test the following hypothesis: a) Copepoda are abundant and diverse at seeps hard substrates and important early colonizers. b) Seepage activity and proximity to foundation species control copepod community structure. c) The community structure, examined at the family level, differs significantly between the two principal substrates analyzed (authigenic rocks and wood blocks).

4.2 Methods

4.2.1 Study area

The Pacific continental margin offshore Central America shows a large number of cold seeps associated with faults, slump scars of submarine landslides, and mounds (Buerk et al., 2010). This is a convergent margin, where one of the most documented geomorphologies is Mound 12 (8° 55.77842’ N, 84° 18.73083’ W) located to the southeast of Nicoya Peninsula.

Mound 12 is an active mud volcano, found at 1020 m water depth, (Niemann et al., 2013), is 30 m high and elongated in northeast-southwest direction with diameters of about 1 to 1.6 km (Mau et al., 2006). At this mound diapirism and mudflows have formed a roundish (~800 m diameter) cone- shaped relief with an irregular pinnacle in the NE and a lower profile ridge in the SW (Niemann et al., 2013). The mound is characterized by the presence of high methane concentrations in the water column, authigenic carbonates, and chemosynthetic communities (Crutchley et al., 2014). Mound 12 seems to be most active at its pinnacle and the SW flank, which is characterized by dense microbial mats and other chemosynthetic organisms (mytilid mussels and Lamellibrachia tubeworms) (Niemann et al., 2013). According to Mau et al. (2006) at Mound 12 bacterial mats are the dominant communities occupied an approximated area of 1500-5000 m2.

The Costa Rica margin has strong vertical hydrographic gradients. Recorded in situ data at Mound 12 in our study showed temperatures of 5.1 °C, Oxygen concentration (by Winkler) between 0.9 and 1.6 L-1, and pH 7.6-7.7 (Levin et al., 2015). Methane output reported by Burkett (2011) in a different study time was 15.5-52.5x103 mol.yr-1, and was considered lower than the reported in other seas (e.g., Norwegian and Mediterranean).

Primary information for this study was obtained during two research expeditions performed by the Scripps Institution of Oceanography -UC San Diego, onboard R/V Atlantis and using the manned submersible Alvin. From the first expedition, cruise AT 15-44 (2009), colonization experiments were deployed. During the second expedition, after 10.5 months, experiments were retrieved from the field (Fig. 4-1), onboard cruise AT 15-59 (2010). Chapter 4 77

A

B C

Figure 4-1: A. Map of the study area is showing the location where substrates were deployed in Mound 12 Costa Rica. B-C. Pictures taken in situ by U. San Diego, USA, during the recovery of experimental substrates: B. Authigenic carbonates and wood blocks; C. Lamellibrach tubes. Background grey regions indicate areas of carbonate cover, orange regions indicate bacterial mats, yellow regions indicated mussel beds or tubeworms clumps. CR=indicate the station number that group together different substrates and habitats combinations. This is used to follow the nomenclature assigned by Grupe (2014). Map based on Grupe (2014). 78 Meio-epifauna Costa Rica

4.2.2 Habitat sampling

Samples were deployed at ~1000 m depth in active and inactive sites on the Costa Rica margin (Table 4-1). Active areas were defined visually by the presence of seepage indicated by bubbling, bacterial mats, and chemosynthetic communities. Inactive sites were selected by the absence of any of the above features. All samples were obtained by a collecting permit issued by the Costa Rica Ministerio de Ambiente y Energía, Sistema Nacional de Áreas de Conservación (Levin et al., 2015).

4.2.3 Colonization experiments on Mound 12

This information is part of a larger study proposed by Scripps Institution, conducted to address metacommunity questions about linkages among carbonate, wood and organic substrates for macrofauna, as well as controls on macrofaunal communities in several geomorphologies of Costa Rican margin (Grupe, 2014; Levin et al., 2015). In order to solve questions at different scales, of the entire originally deployed substrates only a subset corresponding to Mound 12 was analyzed in this study. The aim was to carry out small-scale heterogeneity analysis, focused in a target working group on experimental substrates (i.e., copepods) as a proxy for initial colonization processes.

Experimental design. In order to capture the highest diversity of copepods inhabiting active and inactive cold seep areas, four types of substrates (biogenic and non-biogenic) were selected as representative of the hard substrate at this reducing ecosystem: sunken wood falls, authigenic carbonates, and biogenic surfaces as tubeworms or clams.

The colonization substrates were located at six habitats (Table 4-1). In Mound 12 three active (A-) seeps were selected as follow: Mussel beds (A-MB), Tubeworms (A-TW), and Near mussel beds (A-NMB) where methane and sulfide influenced the biological community or geochemical environment (Grupe, 2014). Also were chosen three habitats noted as inactive (I-): Near mussel bed (I-NMB), Rubble (I-RB), and Sediments (I-Sed), without any signs of fluid flow or chemosynthetic fauna (Grupe, 2014). With the exception of NMB, which had both active and inactive sites for rock substrates, all other habitats were nested within a given activity.

Deployed substrates were placed in most of the habitats by duplicate, for a total of twenty-one substrates available for colonization. They were deployed on 22-23 February and 5 March 2009. The substrates were predefaunated or clean (see below) and then placed on the sea-floor. They consisted of authigenic carbonates (six on active areas and five on inactive ones), wood blocks (three active and four inactive), bundles of death tubeworms (one on inactive area) and bundles of clam shells (one on active area) (Table 4-1).

Substrates were retrieved after 10.5 months and placed in an insulated biobox with Plexiglas compartments that maintained separation of fauna. Two carbonate rocks were recovered from each habitat, except in I-Sed site, where only one rock was found. Two wood blocks were collected from each of three habitats, except habitat I-NMB where only one was found, and no woods were analyzed from A-NMB and I-Sed habitat. Biogenic materials were only recovered at A-NMB and I-Sed habitat from active and inactive sites, and represented by one sampled unit respectively. Chapter 4 79

Prior processing of substrates. Carbonate rocks, previously collected by submersible from methane seeps at Hydrate Ridge, Oregon (44°40’N, 125°6’W) or from Mound 12, were dried in the air for about a week and then deployed. To calculate surface area, these rocks were covered with a single layer of aluminum foil, which was later weighed, and surface area calculated given the mass of a 5 x 5 cm foil square. Untreated Douglas fir wood was cut into blocks approximately 9 x 9 x 24.6 cm, for a total surface of 1047.1 cm2. Biogenic materials were arranged into clumps. Their surface areas were calculated using the measured dimensions of polychaete tubes or clam shells and the formulas for common geometric shapes. All substrates surface areas are given in Table 4-1. Each substrate piece or clump was bagged with 1.6 cm polypropylene mesh and attached a floating polypropylene loop to aid handling, and a lead weight added to wood blocks to ensure sinking.

4.2.4 Density quantification

On board, samples were washed with water, and the liquid residual was sieved through 300 and 43 µm mesh to recover meio-epifauna. The meio-epifauna fraction was fixed in 8 % buffered formalin. In this chapter only copepods were considered; the other faunal groups are addressed in other chapters or ongoing studies. To extract specimens, meio-epifauna was stained with Rose Bengal, and the sample fraction retained by a 43 µm mesh net was added to Ludox HS 40 (density 1.31 g cm-3) for density decantation extraction (5-6 times) follow D. Leduc-Meioscool (2013).

Copepods were separated from the bulk of meio-epifauna by manual picking. They were sorted and counted under Carl Zeiss Discovery V8 and V12 stereo microscopes and identified under light microscope Leica DMi1. Sorted material was preserved in 70 % ethanol. Copepods found in each type of substrate were separated into vials. Following Frontier (1981) and De Oliveira-Diaz et al. (2010) references, a subsample was taken, i.e., between 100 and 500 individuals were counted and identified. Juvenile copepods were counted in a copepodid stage subcategory. All abundances were standardized to 100 % of total sample.

Adults were identified to family level, using standard morphological characteristics following Boxshall & Hasley (2004), and Razouls et al. (2017), among others. Studies at higher taxonomic categories (family) in meiofauna show this level is informative to describe assemblage structure (Herman & Heip, 1988). This rank was used instead of lower ones, as the high probability of finding new genera and species in extreme environments (Gheerardyn et al., 2009; Plum et al., 2015, 2017), might increase the taxonomic effort substantially.

Table 4-1: Habitat, survey dates, and depths of substrates deployed at 2009. A manned submersible recovered experimental substrates after ~317 days of deployment at Mound 12, Costa Rica. Other common characteristics to Mound 12 in this study: Temperature 5.1 °C, Oxygen (Winkler) 0.9-1.6 L-1, pH 7.6-7.7 (Levin et al., 2015). Act.=Seepage activity, A=Active, I=Inactive.

Station Alvin Habitat Act. Substrate Date Date Depth Latitude N Longitude W Surface dive type and deployed recovered (m) area cm2 number CR1 4586 Near mussel bed I Wood# 2 Feb-22-09 Jan-07-10 997 8°55.83484’ 84° 18.75482’ 1047.1 4501 Near mussel bed I Rock# 2 Feb-22-09 Jan-07-10 997 8° 55.83484’ 84° 18.75482’ 599.8 4501 Near mussel bed I Rock# 4 Feb-22-09 Jan-07-10 997 8° 55.83484’ 84° 18.75482’ 825.6 CR2 4587 Mussel bed A Wood# 1 Feb-2209 Jan-08-10 997 8° 55.83538’ 84° 18.75591’ 1047.1 4586 Mussel bed A Wood# 4 Feb-22-09 Jan-07-10 997 8° 55.83538’ 84° 18.75591’ 1047.1 4501 Mussel bed A Rock# 1 Feb-22-09 Jan-07-10 997 8° 55.83538’ 84° 18.75591’ 561.5 4501 Mussel bed A Rock# 3 Feb-22-09 Jan-07-10 997 8° 55.83538’ 84° 18.75591’ 740.2 CR3 4588 Tubeworms A Wood# 5 Feb-23-09 Jan-09-10 995 8° 55.78981’ 84° 18.74010’ 1047.1 4588 Tubeworms A Wood# 6 Feb-23-09 Jan-09-10 995 8° 55.78981’ 84° 18.74010’ 1047.1 4502 Tubeworms A Rock# 5 Feb-23-09 Jan-09-10 995 8° 55.78981’ 84° 18.74010’ 1044.1 4502 Tubeworms A Rock# 6 Feb-23-09 Jan-09-10 995 8° 55.78981’ 84° 18.74010’ 1000.9 CR4 4588 Rubble I Wood# 7 Feb-23-09 Jan-09-10 997 8° 55.79524’ 84° 18.75482’ 1047.1 4589 Rubble I Wood# 8 Feb-23-09 Jan-10-10 997 8° 55.79524’ 84° 18.75482’ 1047.1 4502 Rubble I Rock# 7 Feb-23-09 Jan-09-10 997 8° 55.79524’ 84° 18.75482’ 926.2 4502 Rubble I Rock# 8 Feb-23-09 Jan-10-10 997 8° 55.79524’ 84° 18.75482’ 1371.7 CR5 4511 Near mussel bed A Rock# 9 Mar-05-09 Jan-10-10 1000 8° 55.83376’ 84° 18.77118’ 609.3 4511 Near mussel bed A Rock# 10 Mar-05-09 Jan-10-10 1000 8° 55.83376’ 84° 18.77118’ 788.0 4511 Near mussel bed A Clam shells# Mar-05-09 Jan-10-10 1000 8° 55.83376’ 84° 18.77118’ 585.0 1 CR6 4511 Sediments I Rock# 11 Mar-05-09 Jan-08-10 1000 8° 55.85926’ 84° 18.77336’ 697.9 4511 Sediments I Lamellibrach Mar-05-09 Jan-08-10 1000 8° 55.85926’ 84° 18.77336’ 1914.0 tubes# 2 4511 Sediments I Clam shells# Mar-05-09 Jan-08-10 1000 8° 55.85926’ 84° 18.77336’ 468 2

4.2.5 Data analyses

Meio-epifaunal copepod families were identified, quantified, and data were organized in a matrix of abundance per family in each sample, with habitat, seepage activity and substrate labels. Copepod total density was standardized to ind.10 cm-2. Family mean density for seepage activity (active, inactive), habitat (mussel beds, tubeworm bushes, etc.), and each substrate type (authigenic rocks, block woods, etc.) was calculated for descriptive purposes.

Experiment analyses. To determine statistical differences related to seepage activity on a broad scale, regardless of habitat or substrate, a Bray-Curtis dissimilarity resemblance matrix (fourth-root transformed – densities by added substrates), was used to perform a one-way ANOSIM, with a 9999 permutation, with a p< 0.05 significance level. This tested the following hypothesis: There was no differences in the composition and density of meio-epifaunal Copepoda between seepage activity. As differences were found, a SIMPER analysis was performed.

On a small-scale, the possible effect of the different habitats and substrates in structuring the assemblage was then analyzed. To determine differences in copepod assemblage between habitats (NMB, MB, Sed, TW, RB) and between substrates (wood blocks, authigenic rocks), an one-way ANOSIM was performed for each factor separately. As differences between habitats were found, a

SIMPER analysis was performed. In the first analysis, the null hypothesis Ho1 is that there are no differences in the composition and density of copepod families among the habitats studied, regardless of substrate and activity; in the second, null hypothesis Ho2 is that there are no differences in the composition and abundance of copepod families between wood and rock substrates, regardless of activity and habitat. Then, a two-way nested ANOSIM (habitats nested within activities) was carried out to compare activities and habitats simultaneously. For each test the Global R statistic was calculated independently. In these tests, to avoid pseudo-replication and bias for differences in sample size among substrates, replicate substrates within a given habitat were averaged in a single value, resulting in 10 substrate sample units. Clams and tube substrates were not included in these analyses, and only were used descriptively.

Copepod spatial community structure. To compare activity-habitat combinations in a spatial dimension (=stations of Fig. 4-1) according to the background where they were located (within and outside hardgrounds, cf. Mau, 2004 and Grupe, 2004), another two-way nested ANOSIM was carried out on overall family density means in each of the six habitats studied. This averaging was done to avoid pseudo-replication due to the proximity of each substrate (Fig. 4-1), as well high mobility of copepods, resulting in 6 sample units. Comparisons were done between habitats on hard grounds (I- NMB, A-MB, and A-NMB) and outside hard grounds (A-TW, I-RB and I-Sed), regardless of activity (see Fig 4-1). These combinations correspond to the stations located in Fig- 4-1, and hence they will be called stations. Then, multivariate and univariate analyses were performed on these six stations to describe the spatial structure of the copepod assemblages in Mound 12. A Cluster and an MDS were performed from the Bray Curtis similarity matrix. Densities in 10 cm-2 area were fourth root transformed (to down-weigh high abundance families), for this analysis all types of substrates were used, i.e. rocks, woods, clams and tubes. According to the clusters obtained, a Similarity Profile Analysis (SIMPROF) was carried out to determine significant cluster structure. Similarity Percentage (SIMPER) analysis was next performed in order to determine which families were

82 Meio-epifauna Costa Rica responsible for the clusters. SIMPER analysis was contrasted with BEST analysis to determine which families are responsible for explaining the community's patterns (by maximizing a rank correlation between their respective resemblance matrices) (Clarke & Gorley, 2015). These analyses were performed using PRIMER (Plymouth Routines in Multivariate Ecological Research) v. 7.0 programme (Clarke & Gorley, 2015).

Finally, different univariate ecological attributes were combined and classified them into four categories (rarity, heterogeneity, equitability, and taxonomic diversity) using Guisande et al. (2014) procedure that allows finding differences between the different assemblages studied. In the present study, the Rarity Index of Guisande et al. (2014), Simpson's dominance (Simpson, 1949), Pielou's equitability (Pielou, 1966), and Taxonomic distinctness (Clarke & Warwick, 1998) were selected. The graph obtained is a representation of polar coordinates in which two more ecological attributes can be visually superimposed; in the present case were the number of families and Shannon-Weaver diversity (Shannon & Weaver, 1949).

4.3 Results

4.3.1 Faunistic variables

Of all habitats and substrates surveyed on Mound 12, the most prominent taxon within meio-epifauna was Copepoda, with 73.3 % of total abundance. Nematoda, Polychaeta, Mollusca, and other taxa accounted for the 26.7 % remaining. This work will only focus on copepods; other meio-epifaunal components correspond to unpublished data (see Chapters 2 and 5). After 10.5 months of colonization of different substrates recovered, a total of 24,279 individuals of Copepoda were obtained (Fig. 4-2, Table 4-2), but Clams#2 sample was not added to the total number of individuals presented in the table, as it was not possible to analyze it at the family level. However, it is already shown as it represents an important data to highlight the higher densities in inactive areas.

Of the above, 498 were in copepodite stage, 1,881 were ovate females, and 2,532 belonged to nauplii stage (Fig. 4-2). As the copepods were the dominant group within the crustaceans, it is assumed that a large part of the nauplii larvae belongs to them.

Copepods of the orders Harpacticoida, Siphonostomatoida, Cyclopoida, Poecilostomatoida, and Calanoida orders were present, for a total of 15 families (Table 4-2). The contribution (%) of different families to the total copepod meio-epifauna is shown in Table 4-2. The order Harpacticoida was the best-represented regarding families and densities. Miraciidae (38.6 %), Ectinosomatidae (34.2 %), Tegastidae (9.4 %) and Ameiridae (4.9 %), accounted for approximately 87.1 % of all copepods density, while Dirivultidae (Order Siphonostomatoida) added 7.3 % (Table 4-2). Nauplii larvae were found in all substrates with variable abundances (Table 4-2, Fig. 4-3).

Seepage activity (active vs. inactive). According to seepage activity, 13 families were present respectively. A turnover in family dominance between Miraciidae and Ectinosomatidae, according to seepage activity, was observed. Family Miraciidae accounted for the highest densities at inactives sites, while Ectinosomatiidae had the highest densities at active sites (Fig. 4-2). One-way ANOSIM demonstrated differences between seepage activities (Table 4-3, test 1). A global R=0.52 (p=0.008) Chapter 4 83 indicates that activity does have an effect on assemble structure. SIMPER Analysis (Table 4-4) showed that in Active sites (similarity of 63.5 %) the Ectinosomatidae family contributes 35.0 % while in Inactive sites (average similarity 73.8 %) family Miraciidae contributes with 28.8 % in the configuration of differences. The results also showed that more ovated females were observed in inactive sites than active ones, in a 5:1 ratio (Table 4-2).

Habitat within seepage activity. Mean density shows a pattern between families Miraciidae and Ectinosomatidae according seepage activity and habitat, the former being more representative in inactive sites characterized by the presence of sediments, while Ectinosomatidae was more representative in active sites with the presence of foundation species such as TW and MB (Fig. 4-4). One-way ANOSIM test (Table 4-3, test 2) showed significant differences between habitats, Global R=0.707, p=0.004. The habitats that showed differences and the families that were responsible for those were indicated in Table 4-4 (test 2); In common, it was found that the main families that contributed to the dissimilarities were Cyclopidae, Dirivultidae, Miraciidae, Tegastidae and Ectinosomatidae.

By habitat, taxonomic richness, defined as the number of different families present among them, varied, with the highest values at I-RB site with 12 families (n=4 substrates) followed by A-MB site with 11 (n=4), A-TW site with 10 (n=4), I-Sed site with 10 (n=2), I-NMB site with 8 (n=3) and A- NMB site with 6 (n=2 substrates) (Fig. 4-4). It is observed among seepage activity, the total highest densities averages (18.6±5.5 ind.10cm-2) were found at inactive sites (I-NMB, I-RB, and I-Sed), while in active sites (A-TW, A-MB, and A-NMB) it was 10.5±5.7 ind.10cm-2. Density among families (ind.10cm-2) varied widely (Fig. 4-4); Miraciidae accounted for the highest values in I-NMB (15.6±7.8 ind. 10 cm-2), followed by Ectinosomatidae in A-NMB (13.2±16.5 ind. 10 cm-2).

To compare activities and habitats simultaneously, two-way nested ANOSIM test showed no significant differences between substrates located in active vs. inactive sites using habitat groups as samples (global R=0.611, significant level p=0.1) while a strong variation was found in habitats within seepage activity (global R=0.75, significant level p=0.004) (Table 4-3, test 4).

Inactive sites Active sites

100% Copepodite 90% Calanidae Clausocalanidae 80% Eucalanidae Oncaeidae 70% Corycaeidae 60% Cyclopidae Cyclopinidae 50% Dirivultidae Peltidiidae 40% Cletodidae

Relative abundance % abundance Relative 30% Aegisthidae Ameiridae 20% Miraciidae Tegastidae 10% Ectinosomatidae 0%

B C D Figure 4-2: A. Relative abundance (%) by individual substrate of different copepod meio-epifaunal families in active and inactive sites, and habitats. The most representative families are illustrated: B. Miraciidae, C. Ectinosomatidae, and D. Tegastidae. A=Active, I=Inactive, MB=Mussel beds, NMB=Near Mussel Beds, RB=Rubble, Sed=Sediments, TW=Tubeworms. Type of substrates: C=Clams, R=Rocks, T=Tubes, W=Woods.

Chapter 4 87

Inactive sites Active sites

100%

90%

80%

70%

60%

50%

40%

30% Relative abundance (%) abundanceRelative 20%

10%

0%

Adult Juvenile Nauplii

Figure 4-3: Relative abundance (%) of copepod stages found in the meio-epifauna colonizing different substrates in active and inactive habitats at Mound 12. A=Active, BM=Bacterial mats, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, Sed=Sediments, TW=Tubeworms. Type of substrates: C=Clams, R=Rocks, T=Tubes, W=Wood.

Table 4-3: Results of the various ANOSIM analyses comparing composition and density of meio-epifaunal copepod families colonizing for 10.5 months experimental substrates (Clams and tube substrates were not included in these analyses, only rocks and woods) located in six seepage active and inactive habitats and according to the background where they were located (inside and outside carbonate grounds). A=Active, BM=Bacterial mats, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, Sed=Sediments, TW=Tubeworms. *=significant.

COPEPOD COLONIZATION Sample Permutation Significance level statistic used ANOSIM test 1 (10 samples) (R) Global test Seepage activity 0.52 126 0.8 % (p=0.008)* ANOSIM test 2 (10 samples) Global test Habitat 0.707 4725 0.4 % (p=0.004)* I-NMB. I-RB 0.75 3 33.3 I-NMB. I-Sed 1 3 33.3 I-NMB. A-MB 1 3 33.3 I-NMB. A-TW 0.25 3 66.7 I-NMB. A-NMB 1 3 33.3 I-RB. I-Sed 1 3 33.3 I-RB. A-MB 1 3 33.3 I-RB. A-TW 0 3 66.7 I-RB. A-NMB 1 3 33.3 I-Sed. A-MB 1 3 33.3 I-Sed. A-TW -1 3 100 A-MB. A-TW 0.5 3 33.3 A-MB. A-NMB 1 3 33.3 A-TW. A-NMB 1 3 33.3 ANOSIM test 3 (10 samples) Global test Substrate -0.222 210 95.2 % (p=0.952) ANOSIM test 4 (10 samples) Global test Two-Way Nested (B within A) - B(A). A=Activity, B=Habitat Tests for differences between unordered Activity 0.611 10 10 % (p=0.1) groups (using Habitat groups as samples) Tests for differences between unordered Habitat 0.75 225 0.4 % (p=0.004)* groups (across all Activity groups) ANOSIM test 5 (6 samples) Global test Tests for differences between activity groups (across 1 9 11.1 % (p=0.111) all Background groups) Tests for differences between background groups 0.25 33.3 % (p=0.333) (using Activity groups as samples)

Chapter 4 89

Table 4-4: Results of the SIMPER analyses showing copepod families contributing most to the similarity in activity (A=Active, I=Inactive), and habitats (Group I-Sed and Group A-NMB less than 2 samples in group). Groups were ranked according to their average similarities (Av. sim.) and families according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given.

Contrib. Cum. Families by group Av. Abund Av. Sim Sim/SD (%) (%) SIMPER test 1 Seepage activity groupsGroup A Average similarity=63.49 Ectinosomatidae 1.46 22.09 5.25 35.0 35.0 Miraciidae 0.86 13.39 4.30 21.1 56.1 Dirivultidae 0.69 9.58 2.81 15.1 71.1 Group I Average similarity=73.79 Miraciidae 1.74 21.28 5.61 28.8 28.8 Ectinosomatidae 1.21 14.90 7.33 20.2 49.0 Tegastidae 1.11 13.29 5.98 18.0 67.0 Ameiridae 0.98 12.47 7.36 16.9 83.9 Groups I & A Average dissimilarity=40.09 Group I Group A Contrib. Cum. Av. Abund Av. Abund Av. Diss Diss/SD (%) (%) Miraciidae 1.74 0.86 6.94 2.44 17.3 17.3 Tegastidae 1.11 0.57 4.90 1.43 12.2 29.5 Dirivultidae 0.82 0.69 4.07 1.16 10.2 39.7 Ameiridae 0.98 0.47 3.86 1.58 9.6 49.3 Cyclopinidae 0.47 0.39 3.34 1.25 8.3 57.6 Ectinosomatidae 1.21 1.46 2.87 1.18 7.2 64.8 Cyclopidae 0.32 0.00 2.35 1.18 5.8 70.6 Contrib. Cum. SIMPER test 2 Av. Abund Av. Sim Sim/SD (%) (%) Habitat groups Group I-NMB Average similarity=74.68 Miraciidae 1.99 29.97 40.14 40.14 Ectinosomatidae 1.26 16.7 22.36 62.5 Ameiridae 1.05 15.01 20.1 82.6 Group I-RB Average similarity=80.27 Miraciidae 1.54 17.41 21.69 21.69 Ectinosomatidae 1.26 15.42 19.22 40.9 Tegastidae 1.09 12.75 15.89 56.79 Ameiridae 0.9 11.17 13.92 70.71 Group A-MB Average similarity=78.09 Ectinosomatidae 1.34 24.32 31.15 31.15 Miraciidae 0.69 9.86 12.62 43.77 Corycaeidae 0.57 8.22 10.53 54.3 Ameiridae 0.38 6.86 8.78 63.08 Eucalanidae 0.45 6.38 8.17 71.25 Group A-TW Average similarity=64.70 Tegastidae 1.14 17.56 27.14 27.14 Ectinosomatidae 1.3 16.31 25.21 52.35 Dirivultidae 0.92 15.43 23.85 76.2 90 Meio-epifauna Costa Rica

Groups I-NMB & I-RB Average dissimilarity=27.15 Group I- Group I-RB NMB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Cyclopidae 0 0.51 3.53 5.68 13 13 Miraciidae 1.99 1.54 3.24 2.17 11.94 24.93 Dirivultidae 0.32 0.74 3.03 1.04 11.15 36.08 Cyclopinidae 0.4 0.4 2.73 0.87 10.06 46.14 Calanidae 0 0.35 2.45 4.03 9.04 55.18 Cletodidae 0.28 0.49 2.08 1.1 7.67 62.85 Eucalanidae 0 0.31 1.99 0.86 7.33 70.18 Groups I-NMB & I-Sed Average dissimilarity=29.07 Group I- Group I-Sed NMB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Dirivultidae 0.32 1.96 11.23 2.53 38.65 38.65 Cyclopidae 0 0.57 3.87 8.21 13.31 51.96 Cyclopinidae 0.4 0.78 2.91 0.74 10 61.96 Tegastidae 0.98 1.41 2.85 3.53 9.81 71.77 Groups I-RB & I-Sed Average dissimilarity=25.16 Group I- Group I-Sed RB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Dirivultidae 0.74 1.96 7.56 6 30.04 30.04 Cletodidae 0.49 0 3 9.05 11.92 41.96 Cyclopinidae 0.4 0.78 2.58 0.73 10.26 52.22 Calanidae 0.35 0 2.17 3.81 8.62 60.84 Tegastidae 1.09 1.41 1.99 2.44 7.89 68.73 Eucalanidae 0.31 0 1.78 0.71 7.07 75.8 Groups I-NMB & A-MB Average dissimilarity=48.11 Group I- Group A-

NMB MB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Miraciidae 1.99 0.69 10.89 4.96 22.63 22.63 Tegastidae 0.98 0.29 5.9 3.11 12.26 34.89 Ameiridae 1.05 0.38 5.63 4.6 11.71 46.59 Corycaeidae 0 0.57 4.79 3.8 9.95 56.54 Eucalanidae 0 0.45 3.78 3.36 7.86 64.41 Cyclopinidae 0.4 0.5 3.42 1.49 7.11 71.52

Groups I-RB & A-MB Average dissimilarity=40.66 Group I- Group A-

RB MB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Miraciidae 1.54 0.69 6.21 4.05 15.27 15.27 Tegastidae 1.09 0.29 5.97 43.74 14.68 29.95 Ameiridae 0.9 0.38 3.86 11.71 9.49 39.45 Cyclopidae 0.51 0 3.79 6.79 9.32 48.76 Cletodidae 0.49 0 3.63 13.24 8.93 57.7 Cyclopinidae 0.4 0.5 3.01 1.51 7.4 65.09 Corycaeidae 0.23 0.57 2.71 1.13 6.67 71.76 Groups I-Sed & A-MB Average dissimilarity =50.78 Chapter 4 91

Group I- Group A-

Sed MB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Dirivultidae 1.96 0.55 10.22 4.52 20.12 20.12 Tegastidae 1.41 0.29 8.13 89.32 16.01 36.14 Miraciidae 1.62 0.69 6.73 4.09 13.26 49.39 Ameiridae 0.99 0.38 4.45 91.61 8.76 58.15 Cyclopidae 0.57 0 4.15 69.62 8.17 66.32 Corycaeidae 0 0.57 4.14 3.49 8.14 74.46 Groups I-NMB & A-TW Average dissimilarity=35.25 Group I- Group A-

NMB TW Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Miraciidae 1.99 1.01 7.98 4.5 22.64 22.64 Dirivultidae 0.32 0.92 5.07 1.4 14.39 37.02 Ameiridae 1.05 0.44 4.76 1.21 13.49 50.51 Cyclopinidae 0.4 0.48 3.79 0.98 10.75 61.26 Ectinosomatidae 1.26 1.3 2.63 1.69 7.47 68.73 Aegisthidae 0.31 0 2.26 0.86 6.42 75.15

Groups I-RB & A-TW Average dissimilarity=31.05 Group I- Group A-

RB TW Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Miraciidae 1.54 1.01 3.75 2.85 12.08 12.08 Cyclopidae 0.51 0 3.67 6.44 11.83 23.91 Cletodidae 0.49 0 3.52 10.55 11.35 35.26 Cyclopinidae 0.4 0.48 3.4 1 10.95 46.21 Ameiridae 0.9 0.44 3.15 0.9 10.15 56.35 Calanidae 0.35 0 2.56 4.27 8.23 64.59 Ectinosomatidae 1.26 1.3 2.37 6.55 7.62 72.21 Groups I-Sed & A-TW Average dissimilarity=31.59 Group I- Group A-

Sed TW Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Dirivultidae 1.96 0.92 7.32 14.92 23.19 23.19 Miraciidae 1.62 1.01 4.33 3.52 13.7 36.88 Cyclopidae 0.57 0 4.03 15.16 12.74 49.63 Ameiridae 0.99 0.44 3.75 0.91 11.86 61.49 Cyclopinidae 0.78 0.48 3.44 1.08 10.9 72.4

Groups A-MB & A-TW Average dissimilarity=37.49 Group A- Group A-

MB TW Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Tegastidae 0.29 1.14 7.45 14.83 19.87 19.87 Cyclopinidae 0.5 0.48 4.2 2.1 11.19 31.06 Ameiridae 0.38 0.44 3.88 4.43 10.36 41.42 Corycaeidae 0.57 0.14 3.73 2.27 9.96 51.38 Dirivultidae 0.55 0.92 3.25 1.51 8.67 60.05 Ectinosomatidae 1.34 1.3 2.91 4.54 7.77 67.82 Miraciidae 0.69 1.01 2.75 1.57 7.34 75.16 Groups I-NMB & A-NMB Average dissimilarity=42.66 92 Meio-epifauna Costa Rica

Group I- Group A-

NMB NMB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Miraciidae 1.99 0.87 10.03 5.59 23.51 23.51 Tegastidae 0.98 0 8.91 3.01 20.89 44.4 Ectinosomatidae 1.26 2.03 7.07 2.24 16.58 60.98 Cyclopinidae 0.4 0 3.17 0.71 7.43 68.41 Dirivultidae 0.32 0.51 3.09 1.12 7.25 75.66 Groups I-RB & A-NMB Average dissimilarity=39.78 Group I- Group A-

RB NMB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Tegastidae 1.09 0 8.62 44.5 21.68 21.68 Ectinosomatidae 1.26 2.03 6.12 5.96 15.39 37.06 Miraciidae 1.54 0.87 5.15 5.2 12.94 50 Cyclopidae 0.51 0 4.03 5.36 10.13 60.13 Cyclopinidae 0.4 0 2.88 0.71 7.24 67.37 Calanidae 0.35 0 2.81 3.48 7.06 74.43 Groups I-Sed & A-NMB Average dissimilarity=52.26 Group I- Group A-

Sed NMB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Dirivultidae 1.96 0.51 11.17 Undefined! 21.38 21.38 Tegastidae 1.41 0 10.83 Undefined! 20.72 42.11 Ectinosomatidae 1 2.03 7.89 Undefined! 15.1 57.2 Cyclopinidae 0.78 0 5.95 Undefined! 11.38 68.58 Miraciidae 1.62 0.87 5.74 Undefined! 10.98 79.57 Groups A-MB & A-NMB Average dissimilarity=38.91 Group A- Group A-

MB NMB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Ectinosomatidae 1.34 2.03 6.77 126.61 17.39 17.39 Corycaeidae 0.57 0 5.61 3.55 14.42 31.81 Cyclopinidae 0.5 0 4.87 1.9 12.52 44.33 Eucalanidae 0.45 0 4.44 3.01 11.4 55.74 Ameiridae 0.38 0.73 3.45 74.25 8.87 64.6 Tegastidae 0.29 0 2.82 8.36 7.24 71.84 Groups A-TW & A-NMB Average dissimilarity=40.07 Group A- Group A-

TW NMB Av.Abund Av.Abund Av.Diss Diss/SD Contrib% Cum.% Tegastidae 1.14 0 10.72 29.9 26.75 26.75 Ectinosomatidae 1.3 2.03 7.11 1.42 17.75 44.5 Cyclopinidae 0.48 0 4.22 0.71 10.53 55.03 Ameiridae 0.44 0.73 3.97 1.13 9.91 64.94 Dirivultidae 0.92 0.51 3.9 11.65 9.72 74.67

Chapter 4 93

Inactive sites Active sites

25 Nauplii Copepodites Calanidae Clausocalanidae 20 Eucalanidae

2 Oncaeidae - Corycaeidae 15 Cyclopidae Cyclopinidae Dirivultidae 10 Peltidiidae

Mean N Ind. 10cm Ind. N Mean Cletodidae Aegisthidae 5 Ameiridae Miraciidae Tegastidae 0 Ectinosomatidae I-NMB I-RB I-Sed A-TW A-MB A-NMB Habitat

Figure 4-4 Mean total density of copepod families found in the different habitats, ordered according to seepage activity. A=Active, I=Inactive, NMB= Near mussel beds, MB=Mussel beds, RB=Rubble, Sed=Sediments, TW=Tubeworms. ANOSIM: Global R=0.707, p=0.004.

Type of substrate. Although the number of deployed units per substrate type was different, the total number of recorded families in each was similar (authigenic rocks=14 families, n=11; wood blocks=13 families, n=7; tubes= 9 families, n= 1), except for clams (only three families, n=1). The total maximum number of individuals (36.4 ind.10 cm-2, without nauplii) per substrate was found in Rock# 9 (active site, NMB habitat), but on average, the different kind of substrates located in inactive sites were those that presented the largest number of individuals (16.4±9.9 ind.10cm-2, n=9), while differente kind of substrates in active sites presented less (8.8±9.9 ind.10cm-2, n=11). On average, densities of 14.5±13.0 ind.10cm-2 (n=11) were found per rock, and 10.2±6.0 ind.10cm-2 (n=7) per wood.

The contribution (Mean ind. 10 cm-2) of different families to copepod meio-epifauna by type of experimental substrate, grouping by woods blocks and by authigenic rocks substrates is shown in Fig. 4-5. By woods blocks and by authigenic rocks substrates, Family Miraciidae was more representative in inactive sites, while in active sites it was Ectinosomatidae (Fig. 4-5). However, one way ANOSIM test showed no significant differences between those substrates (regardless of seepage activity and habitat) (Table 4-3, test 3, Global R=-0.222, p=0.952). The negative value of the Global R could represent the irrelevance of the factor being studied, meaning that substrate is not effectively structuring the copepod community, or because sampling was possibly insufficient.

94 Meio-epifauna Costa Rica

Inactive sites Active sites

25 Nauplii Copepodites Calanidae 20 Clausocalanidae

2 Eucalanidae - Oncaeidae 15 Corycaeidae Cyclopidae Cyclopinidae 10 Dirivultidae

Peltidiidae Mean Mean N. Ind. cm10 Cletodidae

5 Aegisthidae Ameiridae Miraciidae Tegastidae 0 Ectinosomatidae Rocks Wood Rocks Wood Substrates

Figure 4-5: Cumulative density of copepod family composition comparing rock and wood experimental substrates by seepage activity.

4.3.2 Copepod spatial community structure

Assamblage. Multivariate analysis (Cluster and MDS) of the six activity-habitat combinations (substrates combined), which correspond to stations of Fig. 4-1, do not showed a clustering pattern. (Fig. 4-6 A, B). The cluster analysis revealed the presence of a typical configuration (stepped) of assemblages related to environmental gradients (Fig. 4-6 A). That is, a single assemblage with two rather unclear assemblages. The only factor that seems to have an effect on structure is the background where the samples were placed. The group of stations with the closest resemblance (I- Sed, I-RB, and A-TW) were placed in a background which was away from the presence of mussel beds (MB or NMB) habitats or carbonates grounds (as indicated in Fig. 4-1). This group of stations would be less heterogeneous physically and more similar in their composition. This group showed closest similarities but are spatially separated from themselves and from the other three. MDS ordination (Fig. 4-6 B) showed similar results to those of the dendrogram, with an acceptable stress value of 0.01. Nested ANOSIM test 5 (Seepage activity and mussel bed/carbonate ground factors=activity into background) (Table 4-3) showed differences in the copepod composition between active and inactive sites (R=1), but not significant statistically. There is also a slight differentiation by mussel bed/carbonates factor, but it is minimal.

A. Chapter 4 95

Group average Transform: Fourth root Resemblance: S17 Bray-Curtis similarity Activity I-Sed I A

I-RB Outside

carbonate

A-TW

n

o

i

t

a t I-NMB S Carbonate A-MB ground A-NMB

50 60 70 80 90 100 Similarity

B. Non-metric MDS Transform: Fourth root Resemblance: S17 Bray-Curtis similarity

A-MB 2D Stress: 0,01 Similarity 55 75 Activity I A

A-TW

I-RB I-Sed

A-NMB

I-NMB

Figure 4-6: A. Cluster analysis and B. two-dimensional MDS ordination of pairwise Bray-Curtis similarity coefficients of meio-epifaunal copepod family assemblages colonizing for 10.5 months different activity-habitat combinations (=stations of Fig. 4-1). Data were standardized to numbers of individuals per 10 cm2, and square root transformed (means of all substrates in each station). A., there is a single homogeneous group (red lines) from SIMPROF; stations according the presence of carbonated ground are indicated. In B., green and blue lines group clusters produced in A. at the 55 and 75 % similarity threshold. A=Active, BM=Bacterial mats, I=Inactive, MB=Mussel beds, NMB=Near mussel beds, Sed=Sediments, TW=Tubeworms.

BEST analysis results differ from those of SIMPER, because it is not carried out by a-priory defined groups, but with the groups conformed in the multivariate analyses, thus showing which families are 96 Meio-epifauna Costa Rica explaining the assemblage composition. These were Aegisthidae, Ectinosomatidae, Eucalanidae, Miraciidae, and Tegastidae, which presented the highest correlation among each other (Spearman=0.975).

SIMPER analysis results for similarity among stations grouping them a-priory by seepage activity and background as factors, and the families that contributed to similarity of these groups, are shown in Table 4-5. The inactive stations (I), and the non-carbonate background stations (NC) were the most similar, both with an average similarity of 85.6 %, while active stations and carbonate background stations had a lower internal similarity (both with 62.6 % average similarity). This, and a rather low average dissimilarity within groups of each factor (active vs inactive, 30.1 %, carbonate and non-carbonate, 28.4 %), implies that being located in an inactive and non-carbonate site, deployed substrates attract a more homogeneous subset of the copepod families, while being in an active and carbonate site holds a greater variability in copepod family compositions and density. Ectinosomatidae is the family that most influences similarity both in seepage Active (A) and Carbonate background (C) groups. Miraciidae is the family that most influence similarity both in Inactive (I) and No Carbonate background (NC) groups. Also, Miraciidae is the family that most influence dissimilarity in Active & Inactive (A&I) group. Tegastidae is the family that most influences dissimilarity in Carbonate and No Carbonate background (C & NC) group (Table 4-5).

Table 4-5: Results of the Two-Way SIMPER analysis for similarity (using seepage activity and bed/carbonates as factors) and families that contributed to similarity. Stations (=activity-habitat combinations) grouped by seepage activity (A=Active, I=Inactive). Groups were ranked according to their average similarities (Av. sim.) and families according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), and cumulative percentage to similarity (Cum. %) are indicated. C=Carbonate background, NC=No Carbonate background.

Families by group Av. Abund Av. Sim Contrib (%) Cum. (%) Seepage activity groups Group A Average similarity=62.56 Ectinosomatidae 0.87 25.49 40.7 40.7 Miraciidae 0.49 14.03 22.4 63.2 Dirivultidae 0.40 11.09 17.7 80.9 Ameiridae 0.33 7.11 11.4 92.3 Group I Average similarity=85.61 Miraciidae 0.95 17.3 20.2 20.2 Tegastidae 0.62 12.53 14.6 34.8 Ectinosomatidae 0.69 12.47 14.6 49.4 Ameiridae 0.55 10.2 11.9 61.3 Dirivultidae 0.56 8.5 9.9 71.2 Cyclopinidae 0.39 7.58 8.9 80.1 Cyclopidae 0.19 5.57 6.5 86.6 Eucalanidae 0.17 4.19 4.9 91.5 Groups A & I Average dissimilarity=30.1 Group A Group I Av.Abund Av.Abund Av. Sim Contrib % Cum. % Miraciidae 0.49 0.95 6.20 20.6 20.6 Tegastidae 0.27 0.62 3.14 11.3 31.9 Ameiridae 0.33 0.55 2.63 8.7 40.6 Chapter 4 97

Cletodidae 0.05 0.19 2.26 7.5 48.1 Cyclopinidae 0.26 0.39 2.08 6.9 55.0 Ectinosomatidae 0.87 0.69 1.89 6.3 61.3 Corycaeidae 0.16 0.07 1.72 5.7 67.0 Cyclopidae 0 0.19 1.57 5.2 72.2 Aegisthidae 0.06 0.11 1.56 5.2 77.4 Eucalanidae 0.13 0.17 1.54 5.1 82.5 Dirivultidae 0.4 0.56 1.54 5.1 87.6 Oncaeidae 0.18 0.12 1.26 4.2 91.8 Examines Background groups (across all Activity groups) Group C Average similarity=62.56 Av.Abund Av.Sim Contrib % Cum. % Ectinosomatidae 0.86 25.49 40.7 40.7 Miraciidae 0.67 14.03 22.4 63.2 Dirivultidae 0.34 11.09 17.7 80.9 Ameiridae 0.39 7.11 11.4 92.3 Group NC Average similarity=85.6 Av.Abund Av.Sim Contrib % Cum. % Miraciidae 0.77 17.3 20.2 20.2 Tegastidae 0.66 12.53 14.6 34.8 Ectinosomatidae 0.71 12.47 14.6 49.4 Ameiridae 0.5 10.2 11.9 61.3 Dirivultidae 0.62 8.5 9.9 71.2 Cyclopinidae 0.41 7.58 8.9 80.1 Cyclopidae 0.19 5.57 6.5 86.6 Eucalanidae 0.21 4.19 4.9 91.5 Groups C & NC Average dissimilarity=28.4 Group C Group NC Av.Abund Av.Abund Av. Sim Contrib % Cum. % Tegastidae 0.23 0.66 4.78 16.8 16.8 Dirivultidae 0.34 0.62 3.11 10.9 27.7 Aegisthidae 0.17 0 2.36 8.3 36.0 Eucalanidae 0.09 0.21 2.29 8.0 44.1 Miraciidae 0.67 0.77 2.26 7.9 52.0 Cyclopinidae 0.25 0.41 2.23 7.8 59.9 Corycaeidae 0.11 0.11 1.80 6.3 66.2 Ectinosomatidae 0.86 0.71 1.68 5.9 72.1 Cyclopidae 0 0.19 1.52 5.3 77.4 Cletodidae 0.14 0.1 1.33 4.7 82.1 Peltidiidae 0 0.06 1.27 4.4 86.6 Oncaeidae 0.12 0.18 1.24 4.4 90.9

Univariate analysis (Rarity Index, Simpson's dominance, Pielou Uniformity, and Taxonomic Uniformity) were performed (Fig. 4-7). Univariate analysis (using RWizard algorithm) supports what was found in the multivariate analysis. The result shows that CR3 (TW-A), CR4 (RB-I), and CR6 (Sed-I) stations (non-carbonates background stations) were most closely related to the combination of above attributes. 98 Meio-epifauna Costa Rica

Figure 4-7: RWizard algorithm Shannon's diversity is represented in the colour gradient (yellow-red). Circles indicate the number of families. Also given Rarity Index, Simpson's dominance, Pielou Uniformity, and Taxonomic Uniformity.

4.4 Discussion

4.4.1 Faunistic composition

In all substrates located in six habitats on Mound 12, the most prominent taxon was Copepoda. Although this paper is the first to address the Copepoda meio-epifaunal community in deep-sea experiments in Costa Rica cold seeps, previous authors have cited the high occurrence of this taxon in chemosynthetic environments, both in the Atlantic and Pacific Oceans (e.g., Plum et al., 2015). Our results are significant considering that in most studies in extreme environments like hydrothermal vents and cold seeps, nematodes dominate the communities (Baguley et al., 2006; Menzel et al., 2011). Copepods have been recorded in a wide range of hard substrates, so its presence was expected, but not the densities found. The group has been recorded on hard substrates that include microbial biofilms, corals, loose rubble, and sponges, polychaete tubes, seagrass blades, macro algae, mudballs, and artificial substrates, among others (Heip et al., 1985; Thistle & Eckman, 1988; Vanreusel et al., 1997; Atilla et al., 2003; Gobin, 2007; Gheerardyn et al., 2009; Portnova et al., 2014; Cheng et al., 2016; Corgosinho et al., 2016). Copepoda, is in most of that meio-epifaunal studies, the second most abundant and diverse group (e.g., Bright et al., 2010). However, authors like Seifried (2004), Bright et al. (2010), and Gollner et al. (2010) establish that they may even exceed nematodes regarding relative abundance and biomass.

The copepod dominance in the relatively short-term duration of our experiment (10.5 months; see also Chapter 2) suggest these mobile metazoans are pioneers during early stages of succession on Chapter 4 99 hard substrates of the deep sea. Nematodes, in contrast, seem to dominate the developed meio- epifaunal communityof the natural carbonate rocks in Costa Rican cold seeps (Chapter 2). Although other studies to compare the dominance of copepods on cold seep hard substrates are unknown, on natural shallow environments, the first settlers were copepods, i.e. cyclopoids, dominating all sampled temporary ponds (Frisch & Green, 2007). The latter author suggests that fast dispersal and dominance of certain cyclopoid copepods during early colonization is related to their ability to store sperm and fast individual development. This could indicate that, at least in shallow waters, environmental conditions would not be determining colonization, but rather the group's own biological characteristics. Also, harpacticoids colonized experimental submerged shallow platforms within days (see Coull, 1988 review). Studying species assemblages of benthic harpacticoid copepods on tide rock pool seaweeds it was found that harpacticoid copepods represented 46.1 % of total meiofauna in the sediment adjacent to Ulva reticulata while only 26.6 % was found inhabiting the seaweed (Zaleha et al., 2010). Fonsêca-Genevois et al. (2006) found, also for shallow waters but in bare aluminium surfaces, that harpacticoid copepods were the fastest meiofaunal colonizers, consistent with their comparatively high propensity to emerge from sediments and colonize a variety of artificial substrates. Although in such experiment the first nematodes appeared on the plates after two days, they reached maximum densities later than did copepod populations. Thus, it is not unlikely that copepods are the first colonizers also in the deep sea.

At the order level, the most dominant group was harpacticoids. Its dominance was expected as this order is characteristically benthic. Harpacticoids appear not to be restricted by geographical barriers such as ridges, long distances or large land masses (Menzel et al., 2011). They are characterized by their small size (body length typically less than 1 mm), and high abundance, and diversity in the deep sea (Thistle & Eckman, 1988; Baguley et al., 2006). Harpacticoids feeding spectrum is broad, including microalgae, protozoans, bacteria, and organic detritus particles (Buffan-Dubau et al., 1996), therefore, they would not be limited in this aspect in Mound 12. This group exhibit differences in lifestyle from infaunal burrowers to epifaunal walkers (Thistle & Eckman, 1988).

Harpacticoids have also been shown to have morphological adaptations to the deep-sea environments. Montagna (1982) found that lengthening and narrowing of most morphological features (in particular the antennule, caudal rami, and the proportion of total body length) are linearly related to increasing depth. In deep sea, harpacticoids have the ability to inhabit multiple habitats (Baguley et al., 2006), and some, according to their shape, are best represented in hard substrates. Several shallow harpacticoids have features specialized to live in substrates other than sediment grains; they may have various body and leg shapes that serve for example to clinging to the substrate (Zaleha et al., 2010). Some families, such as Tegastidae, have been noted for its association with artificial and hard substrates, owing to a body shape that might not be suitable to live interstitiallly (Gollner et al., 2008). The latter coincides with a high frequency of occurrence of this family in our colonization substrates (Table 4-2). Of the two families that were dominant in the Mound 12 community, Ectinosomatidae is characterized by a wide range of features such as their body spindle- shaped (e.g., torpedo-shaped or vermiform), by being epi- and endo-benthic, ubiquitous, typical opportunists, with benthic and phytal species; while Miraciidae has an elongated body, somewhat tapering towards the end, with mostly benthic but also phytal and pelagic species; both families occurr worldwide from littoral to deep-sea bottoms (Giere, 2009). 100 Meio-epifauna Costa Rica

The copepod fauna found in the studied substrates in Costa Rica was also similar in terms of total abundance and family-level composition to that reported in copepod colonization of organic and inorganic substrates by Plum et al. (2017) in Atlantic hydrothermal vents. In our study we found 5 orders, 15 families and a total abundance of 24,279 individuals of Copepoda, while Plum et al. (2017) reported 5 different orders with Harpacticoida being the most common, and 15 families among a total of 26,021 identified individuals. In the latter study, the most species-rich families of harpacticoids were Ameiridae, Ectinosomatidae, and Miraciidae, while those with the highest densities were Ameiridae, Tegastidae, and Tisbidae. These families were also dominant in our study (Table 4-2), with Miraciidae accounted the 38% of density. Furthermore, in Juan de Fuca Ridge (Pacific Ocean), Tsurumi et al. (2003) found on vestimentiferan tubes surfaces and chimney surfaces copepods belonging also to five orders with siphonostomes being the most common, followed by harpacticoids. These authors found that their abundance was highly variable (between 10,000 and 39,800 specimens), and upper-density estimates were about 0.5 copepod cm-2. In the early stage of colonization studied at Mound 12 a reason that should be explored to explain the high numbers of specimens found could be associated to the lack of biotic interactions, as copepods apparently did not have high predation or competition. As observed in macrofauna, most of the organisms present in the substrates were gastropods (Grupe, 2014), and most of these are considered bacterial grazers. In experimental colonization experiments on vents, carried out with inorganic (slate as a proxy for basalt) and organic (wood) substratess deployed on three different localities up and around the Eiffel Tower edifice, groups such as Cyclopoida (Cyclopinidae), Harpacticoida (Ameiridae, Ancorabolidae, Ectinosomatidae, Laophontidae, Miraciidae, Pseudotachidiidae, Donsiellinae, Tegastidae, Tisbidae), and Siphonostomatoida (Dirivultidae) were recorded, with Tisbidae restricted to wood panels (Cuvelier et al., 2014). The latter could indicate that some groups have preference for substrate; in the case of wood, their short-term degradation and associated chemistry could be important. In shallow waters, some similar results were observed, but with lower abundances, only 700 individuals belonging to 14 families, where Miraciidae (21 %), Parastenheliidae (15 %), Ameiridae (13 %) and Laophontidae (13 %) were the dominant families (Sarmento et al., 2012).

Mound 12 oxygen concentration ranges from 0.9-1.6 L-1 (Chapter 2, Appendix 1), which indicates that the values are low, but not limited. However, it was not possible to obtain data of each of the six habitats. Possibly due to the seepage activity (active vs. inactive sites) this value could vary, and in some way affect the presence or not of some families, and even their densities. Harpacticoid copepods are more sensitive to low oxygen concentrations generally, while nematodes are more tolerant (Grego et al., 2014). On sediments, most harpacticoids are sensitive to a reduced oxygen supply, which restricts their occurrence to the upper sediment layers and favors epibenthic life (Dahms & Qian, 2004).

4.4.2 Community and assemblage’s analysis

Hard background. The composition and intensity of fluid seepage, depth, substrate type, oxygen concentration, biological interactions and hydrographic regime (see Ritt et al., 2010 for references), are the most significant factors known structuring the distribution of seep fauna; some of these factors are strongly correlated. In our study, multivariate and univariate analyses determined that the background of carbonate structures where substrates were deployed, is not important in structuring Chapter 4 101 copepod meio-epifaunal seep community. The results showed that the community is more homogeneous in the habitats (i.e., Sed, RB, and TW) located outside the carbonate and in inactive sites. In our case, the presence or absence of a carbonate background seems not to be important in structuring meio-epifaunal copepods community.

The analysis carried out did not show that the type of substrate was a determining variable in the assemblages (Table 4-3, ANOSIM test 3). It was determined more by larger scale factors such as activity and habitat. Our results also show that seepage activity (active and inactive sites) can also have an influence on copepod community structure, with inactive sites being generally more similar to each other than active sites (Table 4-3, ANOSIM test 1). In Pacific hydrothermal vents it was found that habitat type and vent age appear to determine which copepod species are most abundant (Tsurumi et al., 2003). On the other hand, our study did not allow to explore de effects of depth and geography in structuring the community, as all substrates were deployed in the same depth range (approx. 1000 m), and in the same geographical area.

Substrate. In our study, there were no differences in copepod family composition and abundance between substrates used for the colonization experiment (Table 4-3, ANOSIM test 3). However, some trends according to substrate were observed. Carbonates rocks were on average the more densely colonized substrates. The porous surface of rocks could allow these organisms to hold to the substrate more easily. Cheng et al. (2016) found that cnidarians have more copepod associates than any other group of invertebrates, sustaining a high diversity of copepods with multiple resources, including microhabitats and foods, and one host coral may frequently support more than one species of copepod. Nineteen families of harpacticoid copepods have been recorded associated with the coral degradation zone of Lophelia pertusa reefs in NE Atlantic, and the most species-rich families were Ectinosomatidae (36 species), Ameiridae (29 species) and Argestidae (17 species) (Gheerardyn et al., 2009). The latter could indicate that rock-type substrates are highly suitable for this type of organisms. In our work, average densities of 14.5±13.0 ind. 10 cm-2 were obtained for rock substrates.

In contrast, woods had a lower density than rocks. It was expected that woods pieces, as a more ephemeral substrate than rocks, would show degradation and at the same time high colonization by other drilling invertebrates, which favored cavity building and micro spaces for meio-epifauna. In contrast, wood blocks were almost intact after 10.5 months of submersion (see also Chapter 3); no decomposition process was detected; no galleries or softening were observed, and the initial coloration remained (Fig. 4-2). The low porosity could restrict possibilities for organisms to hold on such substrate. In our work, average densities of 10.2±6.0 ind. 10 cm-2 were obtained for wood substrates. Nevertheless, Gaudron et al. (2010) found that organic substrates were more densely colonized than the carbonate substrate, and copepods were found only in organic substrates: after 1 yr 6.3 ind/dm3 were reported to cold seeps both on wood and alfalfa, and after two weeks 12.5 ind/dm3 were reported to cold seeps on woods.

Otherwise, it has been established that the availability of food is a significant factor in the distribution of organisms in a given environment (Rieper, 1982). In this sense, the type of wood may also be important in structuring sunken wood bacterial communities (Kalenitchenko et al., 2015) and therefore the type of organisms that depend on them for food. Locally enhanced degradation 102 Meio-epifauna Costa Rica processes at these organic falls can lead to reducing conditions and high sulfide concentrations, attracting chemoautotrophic bacteria, both free-living and as symbionts of chemosynthetic fauna (Bienhold et al., 2013). In biogenic substrates, initial colonizers, like bacteria and wood-boring bivalves may provide food sources for a following successional fauna, while at the same time degrading the substrate (Schwabe et al., 2015). Therefore, this is an issue that would be important to address in future studies, i.e., include or analyze microfaunal communities that allow establishing direct relationships with microbial populations. One of the factors that are known to affect the distribution of, e.g., harpacticoid copepods includes the ready availability of food in the form of detrital organic matter or microorganisms such as bacteria, algae, and protozoans (Rieper, 1982). In shallow-water ecosystems, it has been estimated that the major meiofaunal taxa (i.e., nematodes and benthic copepods) graze at a rate of 1 % of the microbial standing stock of both heterotrophs and autotrophs per hour, worldwide (Buffan-Dubau et al., 1996). Laboratory studies have shown that some harpacticoid copepods feed on just about everything from natural food items (i.e., algae, detritus and organic matter on sand grains) to more artificial foods (i.e., tapioca and rice bran, dried bacteria particles, and vegetables). However, in selective feeding experiments, some harpacticoids can distinguish between substrates by the bacteria present, or a microbial film (see Rieper, 1982 for references). From this, some question arises: Are copepods dominant in the early stages of succession in Mound 12 regardless of the type of substrates? Are copepods following a pattern of colonization consistent with the chemical evolution of the substrate?

The replication of organic substrates (tubes and clams shells) in our study was low in comparison to rock and wood substrates to establish any pattern. However, these substrates were well represented by the most dominant families sampled (Table 4-2). The copepod communities associated with tubeworm and mussel aggregations around a hydrocarbon seep (Gulf of Mexico), showed that copepod abundance and biomass were very low among tubeworms and mussels, with 0.22 to 6.08 individuals per 10 cm2 sampled area but, abundance was significantly higher among the mussels; copepod diversity and community composition showed no significant differences between the foundation species (Plum et al., 2015). Given the various studies carried out on organic substrates, a clear pattern is not detectable. Therefore, it is essential to continue with this type of experiments, because in cold seeps areas, these substrates (belonging to foundations species) are those who mainly structure the macro community.

5. Trends in trophic structure and habitat preferences in authigenic cold seep rock meio- epifaunal nematodes at Mounds 11 and 12 of Costa Rica

Abstract

Free-living marine nematodes of the Costa Rican deep cold seep areas in the Pacific are not well known, while nematode fauna of hard-bottom substrates is even less known. Numerical dominance of meio-epifaunal nematodes has been found to occur in assemblages inhabiting natural authigenic carbonates of the Costa Rican continental margin. Nineteen authigenic rocks were retrieved by manned submersible from two close locations (Mounds 11 and 12), each with patchy and localized seepage activity resulting in active or inactive sites, and different habitat characteristics. Genus composition and feeding type of the nematode community were analyzed. The total number of specimens collected was 6,879 in a total mean densitiy of 121.7 ind.10 cm-2. Nematode fauna was represented by two classes, six orders, 17 families, and 27 genera. Family Xyalidae was the most abundant (30.1 %), followed by Comesomatidae (21.2 %) and Linhomoeidae (20.9 %). Daptonema and Metalinhomoeus were the genera with the highest density (29.3 % and 18.2 % respectively). The analyses of nematode assemblage structure demonstrate the existence of slight differences between habitats. Regarding trophic structure, non-selective deposit-feeders were dominant (58 %) followed by epigrowth feeders (29 %). At the family and genus level, the nematode fauna was found to be similar to other hard substrate deep-sea locations of cold seep areas.

Keywords Costa Rica; Hard substrates; Meio-epifauna; Nematodes; Cold seeps; Abundance; Feeding guild

5.1 Introduction

Free-living nematodes are among the most frequent and abundant marine metazoans in benthic environments. Their numerical dominance increases with increasing water depth (up to more than 90 % of the total faunal abundance) (Soetaert & Heip, 1995; Gambi et al., 2003; Bianchellli et al., 2013). These worms are known from terrestrial, freshwater, and marine habitats from shallow to deep-sea habitats (Heip et al., 1985; Zekely et al., 2006a). Nematodes are reported occupying all soft sediment habitats principally, and secondary in lower abundances on biofilms and/or hard substrates

104 Meio-epifauna Costa Rica

(Fonsêca-Genevois et al., 2006; Moens et al., 2014). In aquatic ecosystems, free-living nematodes are characterized by their short biological cycles, very limited active dispersal capacities, lack of pelagic larval stages, survival in extreme conditions, key role within the trophic web, and their high population (Fonsêca-Genevois et al., 2006; Semprucci et al., 2013).

In extreme ecosystems, as hydrothermal vents and cold seeps (Vanreusel et al., 1997; Zekely et al., 2006a), bathyal and abyssal soft bottoms areas (Neira & Decraemer, 2009), sediments in a manganese nodule bottoms (Bassau & Vopel, 1999; Miljuti & Miljutina, 2009; Miljuti & Miljutina, 2011), sunken wood surfaces (Schwabe et al., 2015), deep-sea coral reefs (Raes & Vanrusel, 2005), shelf depths and estuaries (Warwick, 1970), nematodes are also among the most species-rich groups. However, in terms of described number of species, including parasitic taxa, the number is limited (ca. 30,000) (Neres et al., 2010; Moens et al., 2014; Venekey et al., 2014). In abundance, an estimated three-quarter, or more, of all animals on earth are nematodes (Venekey et al., 2014).

As stated above, nematodes may be numerically less important on hard substrates, and copepods may co-dominate, but of all metazoans, only the nematodes are the more ubiquitous. The mechanisms by which they recruit (permanently or temporarily) onto submerged substrates are presumed to be resuspension and passive transport by water currents, but it is unclear whether and how nematodes maintain populations on such substrates (Fonsêca-Genevois et al., 2006).

Nematodes are better known in sedimentary habitats, where they play an essential role in the nutrient mineralization and cycling (Decraemer & Backeljau, 2015). This is explained by their morphological and ecological adaptations. Most of them are long and slender organisms, able thus to move between the sedient grains than to crawl over a large surface (Raes & Vanreusel, 2005). Nonetheless, some families show morphological adaptations to an epifaunal life strategy (Raes & Vanreusel, 2005). On deep-sea dead fragments of the coral Lophelia pertusa there is predominance of nematodes belonging to the epifaunal families Epsilonematidae and Draconematidae. These families are morphologically adapted to “walk” over hard substrates; they have a looper caterpillar-like locomotion using ventral setae (ambulatory setae: Epsilonematidae) or tubes on their posterior body region (posterior adhesion tubes: Draconematidae), sometimes together with tubes on or near the head capsule (cephalic adhesion tubes: Draconematidae) (Raes & Vanreusel, 2005). Other morphological adaptations to extreme conditions could exist. Soetaert (1983 in Heip et al., 1985) found that nematodes were larger (5 times) in shallower areas (175 m) than in deeper ones (305- 1605 m). Also, nematodes present in sulfur-oxidizing conditions exhibit a more slender morphology than oxybiotic species (Jensen, 1987). However, Buck & Berry (1998) found no differences in the relationship between body length and body diameter between seeps and control samples. Also, in shallow water corals some adaptations are present; the dominant species are hard-body nematodes with a stouter body shape, ornamented cuticle, cephalic capsule, somatic setae and developed spinneret plus caudal glands (Armenteros et al., 2012).

In nematodes, a great diversity in buccal structures exists which bears on ecological relationships and the niche of the species (Heip et al., 1985). Obtaining information related to feeding habits in chemosynthetic environments is a challenging task given the diversity and size of taxa involved. At least for nematodes inhabiting sediments, it has been established that despite their similar basic Chapter 5 105 morphology, these occupy very different roles and trophic positions, and this diversity in feeding is reflected in species diversity (Heip et al., 1985).

Nematodes mouth shape and size allows identification of their main food items, which permits their classification into four trophic groups based on Wieser’s classification (Wieser, 1953; Balsamo et al., 2010; Guilini et al., 2012). These trophic groups (Selective deposit feeders, Non-selective deposit feeders, Epistrate-feeders, Predators-omnivores) will be described extensively in the methods section of this document. Although recently other classifications have been proposed, Wieser's is the most widely used and represented in most studies.

In the eastern Pacific, the Costa Rican continental margin has heterogeneous areas with different combinations of reducing systems. Like in other extreme deep-sea environments, these reducing communities support a highly specialized community structured by chemosymbiont-bearing species that could influence microscale diversity throughout supply of substrate, food, refuge, and various biotic interactions. Another conspicuous characteristic of Costa Rican cold seep bottoms is the presence of authigenic carbonates. Their precipitation is driven by the anaerobic oxidation of methane (AOM) which is mediated by consortia of methane-oxidizing archaea and sulfate-reducing bacteria; they are formed as a result of increased alkalinity associated with AOM metabolism (Han et al., 2004; Pierre & Fouquete, 2007; Case et al., 2015). Authigenic carbonates occur in a variety of sizes, morphologies, and mineralogies (Case et al., 2015).

Nematodes are an ideal component of the meio-epifaunal community for investigating among others, abundance, diversify and feeding type patterns in cold-seep habitats. Nematodes are the most abundant y and well-represented members of deep-sea meio-epifaunal communities in natural authigenic carbonates substrates of Costa Rica (Chapter 2 of this document). This meio-epifaunal group exhibit all major feeding strategies and thus different nematodes taxa will benefit from differences in food source and availability. Therefore, in order to better understand authigenic carbonate meio-epifauna inhabiting the Costa Rican continental margin deep sea, this research investigated whether seepage activity and habitat influenced epifaunal nematode community structure and their functional diversity (trophic structure) in two close locations with different environmental conditions (Mounds 11 and 12).

Similar substrates (i.e., authigenic rocks), found in geographically separated two locations (1-2 Km) but with different hydrological characteristics, should be inhabited or colonized by the same set of dominant genera, although the species composition may vary between sites. In this context, this work also aims to test the hypothesis that the genus taxonomic rank assemblage structure of authigenic rock-inhabiting meio-epifauna does not vary significantly at the geographic scale involved (between mounds). Instead, we hypohezised that local seepage activity and particular habitat are more importante in determining community structure and density. 106 Meio-epifauna Costa Rica

5.2 Methods

5.2.1 Study area

The Pacific continental margin offshore Central America shows a large number of cold seeps associated with faults, slump scars of submarine landslides, and mounds (Buerk et al., 2010). For instance, the Costa Rican Margin is characterized by the presence of carbonates, mud volcanoes and gas hydrates (Cordes et al., 2010). The study area is colonized by chemosynthetic communities including Calyptogena clams, Lamellibrachia tubeworms and Beggiatoa bacterial mats (Burkett, 2011). Bacterial mats are the dominant feature (Mau et al., 2006).

B C D

A

Figure 5-1: A. Location of Mounds 11 and 12 cold seeps off Costa Rica continental margin where authigenic rocks were collected. Most abuntant taxa are illustrated: B. Daptonema, C. Metalinhomoheus and D. Dorylaimopsis.

Primary information of this study was obtained during a research expedition performed by the Scripps Institution of Oceanography - UC San Diego, on board R/V Atlantis and using the manned submersible Alvin. From the expedition, cruise AT 15-44, samples (natural rocks) were collected from different associated habitats of cold seeps off Costa Rica (Fig. 5-1).

Mound 11. This low relief mud volcano, about 20 m high (Mau, 2004), is located at 1017 m in depth, and characterized by carbonates, gas hydrates, bacterial mats, and siboglinid tubeworm bushes (Sahling et al., 2008). Gas hydrates and methane are primarily of thermogenic origin (Sahling et al., Chapter 5 107

2008). Bacterial mats is the dominant community, occupying an area of 500-1700 m2 (Mau, 2004). Seepage fluid fluxes at Mound 11 are the highest measured at the continental margin of Costa Rica (Sahling et al., 2008).

Mound 12. This is an active low relief mud volcanoe, 30 m high, located at 1020 m in depth, elongated in northeast-southwest direction with diameters of about 1 to 1.6 km (Mau, 2004, 2006; Niemann et al., 2013). This mound is characterized by the presence of high methane concentrations in the water column, authigenic carbonates, and chemosynthetic communities (Crutchley et al., 2014). Mound 12 seems to be most active at its pinnacle and the SW flank, which is characterized by dense microbial mats and other chemosynthetic organisms (mytilid mussels and Lamellibrachia tubeworms) (Niemann et al., 2013). According to Mau et al. (2006) at Mound 12 bacterial mats is the dominant community, occupying an area of 1500-5000 m2. At this mound benthic fluxes are high in sediments covered by bacterial mats (Sahling et al., 2008).

5.2.2 Collection of natural rocks

From the research expedition, cruise AT 15-44 carried out in 2009, loose carbonate rocks suitable for meio-epifauna (from large cobble to small boulder size), were collected from two locations with different geochemistries, Mound 11 and Mound 12 between 967 and 1,025 m depth (Table 5-1). For the present study, 19 rocks were analyzed. In both locations, active sites were defined by the presence of seepage, indicated by bubbling, bacterial mats, and chemosynthetic communities. Where no signs of fluid flow or chemosynthetic fauna were observed, it was considered an inactive site. In each location rocks were collected in different habitats (Table 5-1). The number of carbonate samples for location, seepage activity and habitat depended on their availability. Natural rocks were collected by the mechanical hand of the submersible and placed into an insulated biobox with Plexiglas compartments that maintained separation of fauna.

5.2.3 Quantification of abundance

On board, carbonate rocks were washed with water, and the liquid residual was sieved through 300 and 43 µm meshes to recover meio-epifauna; big sized nematodes of the macrofauna were also included. The meio-epifauna fraction was fixed in 8 % buffered formalin. To extract specimens, samples were stained with Rose Bengal and added to Ludox HS 40 (density 1.31 g.cm-3) for density decantation extraction (5-6 times), following D. Leduc-Meioscool (2013).

Nematodes found in each substrate were separated from the bulk of meio-epifauna by manual picking, and stored into vials. Nematodes were sorted under a stereo microscope, counted and identified to genus level. Sorted material was preserved in 4 % neutralized formalin. A subsample was taken, i.e., about 100 individuals selected at random, counted and identified per substrate (if 100 specimens were no found, the entire sample was examined). Organisms were transferred to glycerine, mounted on glass slides and identified to genus level. Morphological identifications were carried out using taxonomic guides based on Platt & Warwick (1983), Platt & Warwick (1988), Warwick et al. (1998), and Schmidt-Raesa (2014). Abundances were standardized to 100 % of the total sample. Studies at higher taxonomic categories (genus) in meiofauna show this level is 108 Meio-epifauna Costa Rica informative to distinguish assemblage structure (Herman & Heip, 1988). Additionally, due the high probability of finding nematodes new species (as documented by Zekely et al., 2006b, Gobin, 2007, among others) that category was selected.

5.2.4 Data analyses

Composition and abundance. To calculate carbonate rocks surface area, all rocks pieces were covered with a single layer of aluminum foil, which was later weighed, and surface area calculated given the mass of a 5 x 5 cm foil square. The sample surface area ranged between 188 to 2,174 cm2.

The density was calculated from abundance and substrate surface area. In order to compare among environments and with other meio-epifaunal studies, total abundance was standardized to number of individuals per 10 cm-2 of rock surface area. Data were organized in a matrix of taxa x location, denoting for location seepage activity and habitat and for taxa, family and order, and thropic group (see below).

Community patterns. To describe the structure of the meio-epifaunal assemblages, including locations, seepage activity and habitats, multivariate analyses were performed. A similarity matrix was produced using the Bray-Curtis similarity measure, from fourth root transformed data, to down- weight high abundance groups. The matrix was used for hierarchical agglomerative clustering using group average sorting, and for Multidimensional Scaling Ordination (MDS). All 19 individual substrates were used to establish patterns. According to the clusters obtained, a Similarity Profile Analysis (SIMPROF, Type 1, Significance level 5 %) was carried out to determine significant cluster structure. Similarity Percentage (SIMPER) test (cut-off of 50 %) was next performed in order to determine the contribution of each taxa to the total dissimilarity between each cluster.

To determine the possible significant difference in meio-epifaunal nematodes density due to location, seepage activity, and habitat, a series of one-way analyses of similarity (ANOSIM with 9999 permutations, and a p<0.05 significance level) using the same Bray Curtis resemblance matrix. Also, since habitats were nested in seepage activity (no habitat was present in both active or inactive sites) to simultaneously test for seepage and activity effects, a two-way nested ANOSIM was made. Community structure of meio-epifaunal nematodes colonizing on natural rock substrates found at Mounds 11 and 12 was analyzed to obtain information on genera richness, diversity and evenness. These were measured using Margalef’s (d), Shannon’s (H’), Pielou’s (J’) indices. These analyses were performed using PRIMER (Plymouth Routines in Multivariate Ecological Research) v. 7.0 programme.

5.2.5 Nematode trophic structure

Trophic composition of nematodes was based on mouth morphology. Wieser’s classification (Wieser, 1953) made a division in two groups with four feeding types. Group 1 contains those species in which the buccal cavity is unarmed; Group 2 contains species have an armed buccal cavity. The information of Wieser (1953) classification was complemented also with Moens et al. (2014) and Nasri et al. (2016) as follow: Chapter 5 109

▪ Selective deposit feeders or bacterivores (group 1A), small to minute mouth opening, which feed mostly on prokaryotes. These animals swallow small particles through the suction capability of their esophagus. ▪ Non-selective deposit feeders (group 1B), large unarmed buccal cavity, feeding on organic detritus. These animals exploit a wider array of different sized particles, occasionally including other metazoans. ▪ Epistrate-feeders (group 2A), buccal-cavity with scraping tooth or teeth-epistrate, denticles or other sclerotized structures. Oral cavity allows them to scrape (bacteria, fungi) or suck the food consumed after perforation. Coastal nematodes are assumed to feed this way on benthic diatoms and other microalgae. ▪ Predators-omnivores (group 2B), a buccal cavity with large jaws which can prey upon other meiofauna and on macrofaunal juveniles. The prey is entirely sucked or perforated for sucking out the nutrient liquid.

Information about the feeding mode was defined from direct observations of the specimens mouth parts and supported by the literature.

The Index of Trophic Diversity (ITD) was calculated following Heip et al. (1984) and Heip et al. (1985). It is based on the proportion of each trophic group in each habitat. The partitioning of species over the four main feeding types recognized in nematodes is summarized in this trophic index. The IDT =∑θ2, where θ= the percentage of each feeding type, varying between 0.25 and 1. If ∑θ2 =0.25 represent the highest trophic diversity, i.e., the four trophic guilds account for 25 % each. ∑θ2 =1 indicates that only one trophic type is present, representing a single trophic group marked dominance (lowest trophic diversity, i.e., one trophic guild accounts for 100 % of nematode density) (Heip et al., 1984; Gambi et al., 2003).

Table 5-1: Locations (Mounds 11 ans 12), habitat, survey dates, and depths of substrates collection (natural rocks) in 2009 during AT 15-44 cruise by the Alvin manned submersible at Costa Rica. Act.=Seepage activity, A=Active, I=Inactive.

Location Alvin Habitat Act. Substratum Date Depth Latitude N Longitude W T O2 pH dive number recovered (m) (°C) (mL.L.1)

Mound 11 4504 Bacterial mat A S2 Feb-25-09 1010 8° 54.84256’ 84° 18.22426’ 4.19 1.1-1.3 7.7

4505 Near Clam bed A L2 Feb-26-09 1025 8° 54.84256’ 84° 18.22426’ 4.19 1.1-1.3 7.7

4504 Sediments I S3 Feb-25-09 1007 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

4504 Sediments I S4 Feb-25-09 1009 8° 55.35470’ 84° 18.23789’ 4.19 1.1-1.3 7.7

4504 Sediments I L1 Feb-25-09 1000 8° 55.40516’ 84° 18.24007’ 4.19 1.1-1.3 7.7

4505 Boulder field I S2 Feb-25-09 1020 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

4505 Sediments I S3 Feb-26-09 1019 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

4505 Sediments I S4 Feb-26-09 1019 8° 55.36610’ 84° 18.22917’ 4.19 1.1-1.3 7.7

Mound 12 4501 Mussel bed A S4 Feb-22-09 997 8° 55.78710’ 84° 18.77827’ 5.11 0.99-1.6 7.7

4502 Bacterial mat A S4 Feb-23-09 989 8° 55.77625’ 84° 18.77227’ 5.11 0.99-1.6 7.7

4502 Mussel bed A L2 Feb-23-09 987 8° 55.78601’ 84° 18.64849’ 5.11 0.99-1.6 7.7

4503 Mussel bed A S2 Feb-24-09 990 8° 55.79632’ 84° 18.74228’ 5.11 0.99-1.6 7.7

4503 Tubeworms A L3 Feb-24-09 990 8° 55.79632’ 84° 18.63813’ 5.11 0.99-1.6 7.7

4511 Mussel bed A S1 Mar-05-09 997 8° 55.83972’ 84° 18.75155’ 5.11 0.99-1.6 7.7

4511 Bacterial mat A L1 Mar-05-09 996 8° 55.77733’ 84° 18.77173’ 5.11 0.99-1.6 7.7

4511 Bacterial mat A L2 Mar-05-09 998 8° 55.78764’ 84° 18.64849’ 5.11 0.99-1.6 7.7

4502 Carbonates I S1 Feb-23-09 994 8° 55.78601’ 84° 18.74119’ 5.11 0.99-1.6 7.7

4502 Carbonates I S3 Feb-26-09 987 8° 55.79632’ 84° 18.62723’ 5.11 0.99-1.6 7.7

4503 Sediments I S1 Feb-24-09 967 8° 55.78656’ 84° 18.74174’ 5.11 0.99-1.6 7.7

5.1 Results

5.1.1 Abundance and nematode taxonomic diversity

A total of 6,879 individuals of meio-epifaunal nematodes were recorded, representative of 17 families and 27 genera, associated with authigenic carbonate rocks in two different locations of Costa Rica, between 967 and 1025 m in depth. Total mean meio-epifaunal nematodes density (19 substrates) was 6.4±6.1 ind.10cm-2 (mean±1 SD). A list of taxa and densities are provided in Table 5-2.

The dominant nematode families regarding abundance were the Xyalidae (30.1 %), Comesomatidae (21.2 %), Linhomoeidae (20.9 %), Chromadoridae (6.6 %), and Desmodoridae (5.3 %) (Percentage total abundance for each family ≥5 %). Xyalidae with genus Daptonema was the most abundant group across all of the sampling sites (Fig. 5-2, Table 5-2). The most speciose families were Comesomatidae and Microlaimidae with three taxa respectively (Table 5-2).

The nematodes densities differed strikingly between the two locations. The total highest mean density was found at Mound 11 (11.2±6.2 ind.10 cm-2), and the lowest at Mound 12 (2.9±3.0 ind.10 cm-2).

Inactive Active

100% Theristus spp. Monhysteridae spp.

90% Halomonhystera spp. Aponema spp. Microlaimus spp. 80% Bolbolaimus spp. Epsilonema spp. Draconema spp. 70% Desmoscolex spp. Halichoanolaimus spp. 60% Chromadorita spp. Anticoma spp. Metacomesoma spp. 50% Trissonchulus spp. Dolicholaimus spp.

40% Thalassoalaimus spp. Viscosia spp. Symplocostoma spp. 30% Anoplostoma spp. Anticomidae spp. Terschellingia spp. 20% Nematoda relativeabundande Nematoda (%) Desmodora spp. Chromadoridae spp. 10% Sabatieria spp. Dorylaimopsis spp. Metalinhomoeus spp. 0% Daptonema spp. M11-I-BoulF M12-I-Carb M12-I-Sed M11-A-Bmat M11-A-NCB M12-A-TW M12-A-MB

Figure 5-2: Contribution (%) of nematode genera composition summed for activity-habitat combinations in Mounds 11 and 12 off Costa Rica. A=Active, Bmat=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms.

In both Mound 11 and Mound 12, the same genera were found to dominate the authigenic rock substrates. However, the abundance of these genera were different between the two locations (Table 5-3). Daptonema spp. was the most abundant genus (27.0 % and 35.4 % respectively). The same number of genera was also found in the two localities (25). Margalef’s richness, Shannon Wiener’s diversity (H’), and Pielou’s evenness (J’) indexes had very close values between the two locations studied.

According to the number of taxa per seepage activity, active sites (both Mounds combined) accounted the highest average taxa values (11.5±4.3 taxa), while inactive sites were found 10.7±3.0 taxa (Fig. 5-3 A). Related to density per activity, the means were similar, active sites accounted the highest average values (6.6±7.4 ind.10 cm-2), while the inactive sites were of 6.2±4.8 ind.10 cm-2 (Fig. 5-3 B).

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Figure 5-3: A. Mean number (+1 SD) of of nematode taxa (genera) per seepage activity. B. Mean density of nematode per seepage activity. In both plots, data are combined for Mounds 11 and 12.

Chapter 5 115

According to the activities (active vs. inactive) in each of the locations (Mound 11 vs. Mound 12), composition and relative density showed almost similar patterns. This was more evident on active seepage in both Mounds 11 and 12, where the genera Daptonema (family Xyalidae), Metalinhomoeus (Linhomoeidae) and Dorylaimopsis (Comesomatidae) showed the same trend (Fig. 5-4).

Inactive Active

100% Epsilonema spp. Draconema spp. Viscosia spp. 90% Trissonchulus spp. Metacomesoma spp. Dolicholaimus spp. 80% Microlaimus spp.

Activity Halomonhystera spp. - 70% Theristus spp. Aponema spp. Bolbolaimus spp. 60% Desmoscolex spp. Anticomidae spp. 50% Anticoma spp. Anoplostoma spp. Monhysteridae spp. 40% Halichoanolaimus spp. Symplocostoma spp. Terschellingia spp. 30% Chromadorita spp. Thalassoalaimus spp. 20% Chromadoridae spp.

Nematoda relative abundance (%) abundance relative Nematoda Desmodora spp. Sabatieria spp. 10% Dorylaimopsis spp. Metalinhomoeus spp. 0% Daptonema spp. M11-I M12-I M11-A M12-A

Figure 5-4: Contribution (%) of nematode genera composition summed for activity in Mounds 11 and 12 off Costa Rica. A=Active, I=Inactive, M12=Mound 12, M11=Mound 11.

Based on the number of taxa per habitat, bacterial mats habitat (active site) accounted the highest average number of genera (13.0±7.1), followed by near clam bed (active site) and boulder field (inactive site), both with 12 taxa respectively (although these two habitats have only one sample unit) (Fig. 5-5 A). Relating density per habitat, the means were variable, near clam beds (active site) accounted for the highest values (13.9 ind.10 cm-2), followed by boulder field (inactive site) with 12.4 ind.10 cm-2, bacterial mats (active site) accounted 8.4 ind.10 cm-2 (Fig. 5-5 B).

In our Costa Rican samples, some nematodes were observed covered with bacteria (e.g., Daptonema spp., Dorylaimopsis spp., Sabatieria spp., Metalinhomoeus spp.).

Table 5-3: Univariate community indixes for Costa Rica Mound 11 and 12 nematode assemblages. S=total number of genera, N=Total number of individuals, d=Margalef’s species richness, J’=Pielou’s evenness, H’=Shannon Wiener’s diversity. 116 Meio-epifauna Costa Rica

Locations Diversity indices Dominant genera

S N d J´ H'(loge) Mound 11 25 4,095 2.9 0.7 2.2 Daptonema spp. (27.0 %) Metalinhomoeus spp. (17.5 %) Dorylaimopsis spp. (12.8 %) Sabatieria spp. (8.5 %) Mound 12 25 2,784 3.0 0.6 2.0 Daptonema spp. (35.4 %) Metalinhomoeus spp. (20.3 %) Dorylaimopsis spp. (16.8 %) Sabatieria spp. (3.6 %)

A. 20 18 16 14 12 10 8

Mean N. N. Taxa Mean 6 4 2 0 A-Bmat A-NCB A-MB A-TW I-Carb I-BoulF I-Sed

B.

18 2

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Figure 5-5: A. Mean number (+1 SD) of nematode taxa (genera) per habitat. B. Mean density of nematodes per habitat. In both plots, data are combined for Mounds 11 and 12. M12=Mound 12, M11=Mound 11, A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms. Chapter 5 117

5.1.2 Community patterns

The hierarchical cluster analysis and nonmetric multi-dimensional scaling (MDS) (Figs. 5-6 A, B) from nematode genera composition of the two location (Mound 11 and Mound 12) and several active or inactive habitats, showed a mixed cluster pattern with no consistent clustering or segregation for location, activity or habitats. The SIMPER analysis supported four main groups (A, B, C and D), with two of them (A and B) having a single sample (Table 5-4). Groups C and D show some degree of clustering for location (group D, all from Mound 12) and location-habitat-activity combinations (group C, subgroup M11-I-Sed). Genus Daptonema was the main responsible for similarities between groups. Between groups C (mainly M11 substrates) and D (most M12 substrates) were found an average dissimilarity of 54.9 %, Thalassoalaimus was the main responsible for dissimilarities between groups.

A. Group average Transform: Fourth root Resemblance: S17 Bray-Curtis similarity 20

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118 Meio-epifauna Costa Rica

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2D Stress: 0,19 M12-I-Carb Similarity 47 B 55 M12-A-MB Habitat M12-A-MB D Bmat C NCB TW M12-A-MB MB M11-A-Bmat M12-A-MB M12-A-Bmat BoulF M12-A-Bmat Carb M11-A-NCB Sed M11-I-Sed M12-I-Carb M11-I-SedM11-I-BoulF M12-A-Bmat M12-A-TW M11-I-Sed A

M11-I-Sed

M12-I-Sed M11-I-Sed

Figure 5-6: A. Cluster analysis, and B. Two-dimensional MDS ordination of meio-epifaunal nematodes associated with natural rocks of Mounds 11 and 12 of Costa Rica cold seeps. The main groups produced by a threshold similarity of 47 % are shown. Black lines in the dendrograme indicate groups that are established, red lines showed a substructure from the clustering for which SIMPROF showed no statistical support. A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms.

Table 5-4: Similarity results calculated by SIMPER (19 substrates). Taxa contributing most to the similarity between groups resulting from the cluster analysis. Also shown is the dissimilarity between the two main groups. Groups were ranked according to their average similarities (Av. sim.) and taxa according to their average contribution (Contrib. %) to similarity in each group; average abundance (Av. abund.), similarity/standard deviation (Sim./SD) and cumulative percentage to similarity (Cum. %) are given. Daptonema are the most important contributors to similarity in all groups. Groups A and B less than two samples in group. A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms.

Group C (M11-A-Bmat, M12-A-Bmat, M11-A-NCB, M12-A-MB, M12-A-MB, M11-I-BouldF, all M11-I- Sed) Average similarity=59.1 % Genera Av. Abund Av. Sim Sim./SD Contrib. % Cum. % Daptonema spp. 1.2 9.74 4.55 16.5 16.5 Dorylaimopsis spp. 1.05 9.52 5.14 16.1 32.6 Metalinhomoeus spp. 1.02 7.94 1.94 13.4 46.0 Thalassoalaimus spp. 0.75 6.25 3.45 10.6 56.6 Chapter 5 119

Desmodora spp. 0.72 5.15 1.87 8.7 65.3 Halichoanolaimus spp. 0.54 3.73 1.23 6.3 71.6 Group D (M12-A-Bmat, M12-A-Bmat, M12-A-MB, M12-A-MB, M12-I-Carb, M12-I-Sed) Average similarity=55.46 % Genera Av. Abund Av. Sim Sim/SD Contrib. % Cum. % Daptonema spp. 0.80 14.24 4.30 25.7 25.7 Metalinhomoeus spp. 0.75 13.85 5.68 25.0 50.6 Chromadorita spp. 0.50 9.83 7.85 17.7 68.4 Dorylaimopsis spp. 0.47 6.15 1.29 11.1 79.4 Groups C & D Average dissimilarity=54.91 % Group C Group D Genera Av. Abund Av. Abund Av. Diss Diss/SD Contrib. % Cum. % Thalassoalaimus spp. 0.75 0.14 4.26 1.97 7.7 7.7 Dorylaimopsis spp. 1.05 0.47 3.98 1.82 7.2 15.0 Halichoanolaimus spp. 0.54 0 3.79 1.58 6.9 21.9 Sabatieria spp. 0.55 0.2 3.41 1.19 6.2 28.1 Chromadoridae spp. 0.52 0.16 3.35 1.23 6.1 34.2 Terschellingia spp. 0.5 0.11 3.27 1.18 5.9 40.2 Daptonema spp. 1.2 0.8 3.05 1.82 5.6 45.7 Desmodora spp. 0.72 0.4 2.96 1.25 5.4 51.1 Metalinhomoeus spp. 1.02 0.75 2.84 1.73 5.2 56.3 Chromadorita spp. 0.4 0.5 2.48 1.77 4.5 60.8 Symplocostoma spp. 0.47 0.28 2.39 1.37 4.3 65.2 Desmoscolex spp. 0.35 0 2.31 0.99 4.2 69.4 Anticoma spp. 0.36 0.16 2.14 1.36 4.0 73.3

Across all activities and habitats, the one-way ANOSIM analyses (Table 5-5, test 1) showed significant differences in Nematoda composition and abundance between the two locations analyzed (Mound 11 and Mound 12). Across all locations and habitats, according to the ANOSIM (Table 5-5, test 2) significant differences in community composition and abundances between seepage activities were found. Regarding habitats, ANOSIM test 3 (Table 5-5) showed no significant differences; Two- way nested ANOSIM (Habitat within Seepage activity) also showed no significant differences (Table 5-5, test 4).

120 Meio-epifauna Costa Rica

Table 5-5: Results of the varios ANOSIM analyses comparing composition and density of meio-epifaunal Nematoda between location, seepage activity, and habitat, for natural carbonate rock substrates. A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, M12=Mound 12, M11=Mound 11, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms. *= significant.

Sample Permutation Significance level % statistic (R) used NATURAL CARBONATE ROCKS ANOSIM test 1 (19 samples) Global test Locations 0.3296 9999 0.1 % (p=0.001)* ANOSIM test 2 (19 samples) Global test Seepage activity 0.182 1.8 % (p=0.018)* ANOSIM test 3 (19 samples) Global test Habitat 0.203 6.7 % (p=0.067) Pairwise tests Bmat vs. NCB -0.5 5 100 Bmat vs. TW 0.167 5 40 Bmat vs. MB 0.042 35 40 Bmat vs. BoulF -0.25 5 80 Bmat vs. Carb -0.036 15 66.7 Bmat vs. Sed 0.099 210 21.4 NCB vs. MB -0.25 5 100 NCB vs. Carb 1 3 33.3 NCB vs. Sed 0 7 42.9 TW vs. MB 0.833 5 20 TW vs. Carb 1 3 33.3 TW vs. Sed 0.711 7 14.3 MB vs. BoulF 0.167 5 40 MB vs. Carb 0.357 15 26.7 MB vs. Sed 0.508 210 2.4 BoulF vs. Carb 1 3 33.3 BoulF vs. Sed -0.444 7 100 Carb vs. Sed 0.323 28 17.9 ANOSIM test 4 (19 samples) Global test Two-Way Nested (B within A) - B(A) A=Seepage activity, B=Habitat Tests for differences between unordered Activity groups (using Habitat groups as 0.111 35 25.7 % (p=0.257) samples) Tests for differences between unordered 0.093 9999 28.9 % (p=0.289) Habitat groups (across all Activity groups)

Chapter 5 121

5.1.3 Nematode trophic structure

The nematode community of Mounds 11 and 12 was characterized by non-selective deposit feeders (1B), representing 58 % of nematodes abundance at the two studied sites. This category was followed by epistrate-feeders (2A) with 29 % (Fig. 5-7).

Density contribution nematodes Mounds 11 and 12 feeding types 3% 10%

29%

58%

1A 1B 2A 2B

Figure 5-7: Total proportional abundance of nematode feeding types at Mounds 11 and 12 of Costa Rica. 1A: selective deposit feeders, 1B: non-selective deposit-feeders, 2A: epistrate feeders, 2B: predators and/or omnivores.

According to seepage activity, the non-selective deposit feeders dominated (by density) on active sites with 65 % of the contribution (Fig. 5-8) while in inactive sites they accounted to 50 % of density. This was followed by epistrate-feeders with 28 % of the density in active sites, and 31 % in inactive sites.

100% 90% 80% 70% 60% 50% 40%

Contribution % Contribution 30% 20% 10% 0% Active Inactive 1A 1B 2A 2B

Figure 5-8: Nematode feeding contribution by density per seepage activity (active vs. inactive sites across locations and habitats). 1A: selective deposit feeders, 1B: non-selective deposit-feeders, 2A: epistrate feeders, 2B: predators and/or omnivores. 122 Meio-epifauna Costa Rica

By habitat (Fig. 5-9), the highest contribution of non-selective deposit feeders occurred in bacterial mats (active site) (24.42 ind.10 cm-2), sediments habitat (inactive site) with 17.07 ind.10 cm-2, and mussel bed (active site) with 9.86 ind.10 cm-2. About epistrate-feeders, sediment habitat showed the highest contribution (inactive site) with 13.79 ind.10 cm-2.

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Total N Total 10

5

0 A-Bmat A-NCB A-MB A-TW I-Carb I-BoulF I-Sed 1A 1B 2A 2B

Figure 5-9: Density (total ind.10 cm-2) of nematode feeding groups per habitat (across locations). A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms. 1A=Selective deposit feeders, 1B=Non-selective deposit-feeders, 2A=Epistrate feeders, 2B=Predators and/or omnivores.

The Index of Throphic Diversity (ITD) calculated by habitat showed that the four trophic groups were roughly well represented (none showed a value greater than 0.6), which indicates a relatively high and similar trophic diversity in each habitat (Table 5-6).

Table 5-6: Contribution in abundance (%) of nematode feeding groups and Index of Throphic Diversity (ITD) by habitat (across localities). A=Active, BM=Bacterial mat, BoulF=Boulder Field, Car=Carbonates, I=Inactive, MB=Mussel beds, NCB=Near clam beds, Sed=Sediments, TW=Tubeworms.1A: selective deposit feeders, 1B=non-selective deposit-feeders, 2A=epistrate feeders, 2B=predators and/or omnivores.

Trophic type 1A 1B 2A 2B ITD Habitat A-Bmat 2.2 72.4 24.5 0.9 0.58 A-NCB 8.6 60.7 29.7 1.0 0.46 A-MB 6.6 56.0 32.7 4.7 0.43 A-TW 1.1 48.9 46.7 3.3 0.46 I-Carb 5.6 55.2 37.5 1.6 0.45 I-BoulF 6.7 72.8 18.7 1.8 0.57 I-Sed 19.0 42.2 34.1 4.6 0.33 Chapter 5 123

5.2 Discussion

In the present study, the abundance, composition, and trophic structure of nematodes assemblages, to the genus taxonomic rank, in cold seep authigenic carbonates in Costa Rica, were described. This is the first attempts to characterize community structure of meio-epifaunal nematodes in this deep- sea environments, and the possible factors controlling it. This kind of natural substrate showed a positive influence for the presence of meio-epifaunal groups (see Chapter 2), and in this case for free-living nematodes, in comparison to other substrates such as experimental wood blocks (see Chapter 3). This influence may be due to the architectural and structural complexity of rock surfaces, appropriate for the settlement and dwelling of these organisms. Nevertheless, authigenic carbonates, like any other submerged substrate that is colonized, whether of organic or inorganic origin, may provide the same services. That is, at cm scale, any marine substrates (e.g., branching and filamentous drift and floating algae, branching benthic algal masses, hydrozoan colonies, seagrass epiphytes, sunken woods, rocks, shells, etc) create complex habitats which harbor and support small invertebrates, for critical parts of their life history, and incorporate elements to the food web (Atilla et al., 2005). But in particularly for smaller organisms, authigenic carbonates may provide substrate for settlement and attachment, reproductive sites, refuge from predators and food supply (Levin et al., 2015). The results showed that the nematode community and the feeding types exhibited spatial and habitat differences, but these were not consistent. There is little information in the literature about nematodes in authigenic rocks, so comparisons and analysis will be carried out mainly with cold seeps or hydrothermal vents in other localities that involve inorganic substrates or sediments.

Abundance, composition. In total, 6,879 individuals belonging to 17 families and 27 genera, were found. The four most abundant genera accounted for 68.6 % of the total number of individuals. The dominant genera were Daptonema 29.3 % (Xyalidae), Metalinhomoeus 18.2 % (Linhomoeidae), Dorylaimopsis 13.9 % (Comesomatidae) and Sabateria 17.2 % (Comesomatidae). Xyalidae is a documented inhabitant of nearly all types of marine environments and frequently among the most abundant taxa (Venekey et al., 2014); as will be described below, this family is present in many of the deep-water studies. Worldwide, the family Xyalidae contains 44 described genera (Schmidt- Raesa, 2014), with Daptonema as the genus with the highest number of valid and species inquerendae within the family, having 117 nominal species (Schmidt-Raesa, 2014; Venekey et al., 2014). In our study of carbonate rocks in two different localities (Mound 11 and 12) a similar dominance pattern of genera was found for both active and inactive sites. But it is in active seepage, both to Mounds 11 and 12, where this trend is evident with the genera Daptonema, Metalinhomoeus and Dorylaimopsis. Draconematidae (Draconemma spp.) mentioned as the dominant family in other studies was not highlighted in our study; this family only contributed 0.05% to the total density.

In Hydrate Ridge (offshore Oregon - NE Pacific Ocean), information about nematodes on rocks was included by Sapir et al. (2014). Although the focus of that study was deep-sea parasitic fungi on nematodes, they found abundant nematode assemblages associated with carbonate rocks and sulfide- oxidizing bacterial mats; nematodes comprised 80 % or more of all meiofauna; the most abundant nematode was identified as Desmodora aff. alberti (family Desmodoridae). In our study, Desmodora was less importantly represented on the authigenic rocks, contributing 5.3 % of the total density of nematodes. In this same location, but in sediments a rich nematodes assemblage of 31 families and 124 Meio-epifauna Costa Rica

129 genera was found, where nematode seep communities exhibited low diversity with dominance of only one or two genera (i.e., Daptonema and Metadesmolaimus - Family Xyalidae) (Guilini et al., 2012). The latter study, along with our results, underlines the importance of Xyalidae of the Eastern Pacific cold seeps.

In other basins, several studies that include other hard substrates, showed similar trends in the composition as those found in our study. At Lucky Strike hydrothermal vent field (Mid-Atlantic Ridge) after nine months of colonization on organic and inorganic substrates 24 nematode species belonging to 20 genera and 12 families were found (Zeppilli et al., 2015). In contrast to the results found in our study in the Pacific Ocean, Zeppelli et al. (2015), on slate substrates found Draconematidae as the dominant family. This family, as already mentioned in the introduction of this document, is morphologically adapted to live on hard substrates. In that study, comparisons between substrates found differences in the dominant group. On wood substrate, Monhysteridae was the dominant family, accounting for 42-93 % of the total nematode abundance; whereas bones were largely dominated by Monhysteridae, followed by Microlaimidae and Draconematidae. With genus composition, it was observed that different genera dominated the different substrates, that is Cephalochaetosoma on slate (75-90 %), Halomonhystera on bone (71-92 %), Oncholaimus, on wood. The previous study also included observations after two years of colonization where Draconematidae dominated both on slate and wood, while Draconematidae, Microlaimidae, and Monhysteridae showed co-dominance both on slate and wood. The above could indicate that over time the community could become more heterogeneous.

In shallow waters, Gobin (2007) using artificial substrates (nylon pan scourers) in subtidal hard, rocky bottoms found 25 families, 52 genera, and 70 species. The Chromadoridae were the most abundant followed by the Cyatholaimidae. However, the most abundant species was Cyatholaimus sp. 1 (of the family Cyatholaimidae), contributing to approximately 18 % of the total number of individuals present. Five families: Chromadoridae, Cyatholaimidae, Draconematidae, Oncholaimidae, and Oxystominidae comprised approximately 75 % of the total abundance. Of the above, Draconematidae is highlighted as a typical family of hard substrates, either in shallow or deep water. Similar families and genera were found as in other geographic areas such as New Zealand and the southwest coast of England (Gobin, 2007). The latter reinforces the idea that at least at the family and genus level there are many similarities in the composition of nematode assemblages, whether in shallow or deep water. Further conclusions about changes in the composition of assemblages require information at the species level. This represents a major challenge, given the taxonomic effort in its identification because most species are potentially new to science

For nematodes, studies in other habitats (i.e sediments, both in cold seeps and vents) reinforce the idea of high dominance of some groups that structure the community, these patterns have been found at family, genus and species levels. In the Western Pacific Ocean (Sagami Bay, central Japan) at cold seeps sediments, two conspicuous families were Xyalidae (6 species, 47.2 % abundance) and Chromadoridae (8 species, 20.2 % abundance) with a low number of genera, six and eight respectively; two Daptonema and one Chromadorita species were the dominants, occupying 20.2, 12.9 and 12.3 % of the total nematodes, respectively (Shirayama & Ohta, 1990). On the northern and southern East Pacific Rise, nematode assemblages exhibited high dominance by a few species, with one species of Thalassomonhystera (Family Monhysteridae) most abundant at five of the seven Chapter 5 125 vent sites studied. In that study, the three most abundant nematode species contributed more than 70 % of the total nematode abundance at each site (Copley et al., 2007). In methane seep areas (Norwegian Sea), also in sediments, two studies showed similar results, with a high diversity found. To the Håkon Mosby Mud Volcano, families Chromadoridae (11 species) and Xyalidae (9 species) contained the highest number of species, followed by the Linhomoeidae (7 species) and Diplopeltidae (6 species) (Portnova et al., 2011); while in the Nyegga Region the highest species diversity was also observed in Chromadoridae and Xyalidae families (Portnova et al., 2014). These studies in the same area could indicate that at least in local scales (short distances), colonization patterns in nematodes were similar.

Work at the species level will support or review the conclusions about some nematode cosmopolitan distributions. Shirayama & Ohta (1990) found a little relationship between cold seep nematodes of Japan (Pacific) and the assemblage at a distant place, both with the similar environmental condition. Their conclusions did not support the concept that certain “nematode taxa are specialized to have their life in the thiobiotic condition and have a cosmopolitan distribution. It rather favors the idea that the meiofauna existing in the thiobiotic condition had been originally oxybiotic species which eventually adapted at each place to live in its own local reduced environment”.

Locations, seepage activity and habitats. Statisticall differences by location and seepage activity (across habitats) at Mound 11 and Mound 12 were supported by ANOSIM (Table 5-5, tests 1 and 2), cluster, and MDS (Fig. 5). In our study, accoding an ANOSIM test clear differences were observed between active and non-active sites, with few genera high dominance. But although were found more families and other genera, the same trend in compositon and density contribution were present in both active and inactive sites. Therefore, in order to establish a pattern as indicated by Coopley et al. (2007) analysis at the species level would be required.

About seepage activity and habitat, no general trend is known for deep-sea nematodes. In the NE Pacific Ocean, no significant differences were found between the nematode assemblage composition of clam beds and microbial mats, nor betweeen locations (Guilini et al., 2012). In the Western Pacific Ocean (Sagami Bay, central Japan) at cold seeps sediments was recorded that the meiofauna was slightly (1.5 to 1.6 times) more abundant at the seep site than at the control area (Shirayama & Ohta, 1990). In the North Fiji Basin (western Pacific) hydrothermal vent sediments, it was found that Daptonema (Family Xyalidae) was present in active hydrothermal areas, but its contribution was not very high (range between 0.2 % and 2.4 %), while in the reference areas its contribution was slightly higher (2.1 % and 6.7 %) (Vanreusel et al., 1997). On the other hand, in mud volcanoes of the Norwegian Sea (Portnova et al., 2014) nematode species diversity increased in the direction away from the center of the reducing zone (the bacterial mat), across the siboglinid fields, and to the background biotope, and the highest density was found in background biotope (although these results are not comparative because they are related to meiobenthos).

Zeppilli et al. (2015) study, found some trends according to gas emissions: a) The highest nematode density was reported in wood located at the external site while the lowest was found on wood located at the low emission site, with 242,006.1 and 381.5 ind m−2, respectively. b) In slate substrates, the abundance decreased with decreasing fluid emission, with values 10 times lower at the external site, from 17,641.3 to 1250.0 ind m−2. c) Concerning bones sustrates, the abundance was higher at the 126 Meio-epifauna Costa Rica intermediate site than at the high emission site, 4792.1 and 2807.3 ind m−2, respectively. Trends according to faunal composition and gas emissions are also showed by Zeppilli et al. (2015), Draconematidae increased its dominance with increasing vent emission (from 76 to 93 % of the total nematode abundance, from external to high emission sites, respectively), and Monhysteridae increased its dominance with decreasing vent emission (from 4 to 16 % of the total nematode abundance, from high to low emission sites, respectively). In addition, Microlaimidae was found only on the wood located in the low and external sites, accounting for 20 and 30 % of the total nematode abundance, respectively. The above could suggest that changes in the composition of some taxonomic groups could be influenced by gas emissions intensity (either increase or decrease). Therefore, would be important to measure these emissions in Costa Rica each identified habitat to attempt to establish related patterns.

Although this chapter includes information based on the same type of substrate (authigenic rock), the two locations differ in hydrographic conditions. In terms of depth, both Mound 11 and Mound 12 were in the same range (between 967 and 1025 m depth) so no differences were expected for this factor, but other variables such as temperature and oxygen could play a role in the assemblages structure. However, this needs to be reviewed in the future with specific data on each habitat, as it seems that nematode families, genera and species exhibit a very good tolerance to extreme conditions.

Nematode trophic structure. At Mounds 11 and 12, non-selective depostit feeders (1B) and epistrate feeders (2A) were found to be the predominant trophic groups. Other results involving nematodes of deep-sea and shelf break sites in the North Atlantic and Meditaerranea sea also found bacterial feeders (deposit and epistrate feeders) dominance (Soetaert & Heip, 1995). For subtidal nematode fauna inhabiting experimental hard substrates of Trinidad and Tobago, epistrate feeders were dominant (65.2 %) followed by non-selective deposit-feeders (13.3 %) (Gobin, 2007). In the study area, Beggiatoa bacterial mats are the dominant community (Mau et al., 2006; Burkett, 2011). For hydrothermal vents, it has been documented that most of the meiofauna associated are primary consumers that depend on the food supply from bacterial deposits (i.e., mats or biofilms), resulting in a positive correlation between meiofaunal abundances and the (bacterial) debris (Giere, 2009). Both depostit feeders (1B) and epistrate feeders (2A) trophic groups are known to influence microbial densities by their grazing on bacteria (Jensen, 1987). In cold seeps, “gas emissions are taken up by archaea and bacteria through chemoautotrophic processes to produce sulfides which may be further utilized by symbiotic bacteria to sustain high biomass production in invertebrates” (Lampadariou et al., 2013). Stable carbon isotope analyses also are suggested that bacteria are the origin of chemosynthetically derived food (Van Gaever et al., 2006; Giere, 2009). However, it should also be noted that some other sources of food are available. Natural and artificial substrates found on the bottom capture sediment and other organic matter from the water column, which may provide a food source for some colonizing invertebrates; on the collected rocks the presence of such particles were observed. The results obtained for Mounds 11 and 12 support the above, considering that the feeding groups dominance were non-selective deposit feeders (1B) and epistrate feeders (2A), characterized by feeding upon bacteria, fungi, and organic detritus, among others. As it was found for macrofauna on carbonates, the same diverse sources of organic carbon include surface-derived organic matter, sulfide-oxidizing bacteria, sulfate-reducing bacteria, aerobic methane oxidizing bacteria and anaerobic methane oxidizing archaea at this cold seeps of Costa Rica (Levin et al., Chapter 5 127

2015). It would be important to quantify the relationships between the amount of food and the presence and density of some types of nematodes. The availability of food is especially important for explaining the quantitative distribution of nematodes in the deep sea (Heip et al., 1985).

In our Costa Rican samples, some nematodes were observed covered with bacteria (e.g., Daptonema spp., Dorylaimopsis spp., Sabatieria spp., Metalinhomoeus spp.). This has been observed by other studies that suggest these bacteria may be trapped in some kind of mucus that apparently resisted the elutriation techniques (Soetaert & Heip, 1995). Gardening activity has been documented in some Monhysteridae from the Clarion-Clipperton fracture zone in the deep eastern central Pacific (Renaud-Mornant & Gourbault, 1990 in Soetaert & Heip, 1995). Also in shallow water nematodes, it is suggested that Leptonemella aphanothecae feeds on its external bacteria (Gerlach, 1978 in Jensen, 1987). Probably these bacterial envelopers are reminiscent of “microbial gardens” (Soetaert & Heip, 1995).

According to our ITD, there were no values close to 1 through the diverse habitats, meaning there is no pronounced dominance by a single trophic group. The latter could indicate that there are several sources of food available in these sites other than bacteria and also that Mounds 11 and 12 of Costa Rica support a trophically compex nematode community.

6. Conclusions and recomendations

6.1 Conclusions

This PhD thesis provides a framework for future exploration and biological experimentation of communities associated with organic and inorganic substrates at Costa Rican cold seeps. This extensive environment has received relatively little attention in terms of the knowledge of small fauna, Specifically meio-epifauna living on hard substrates have been understudied. Knowledge on the colonization patterns of these meio-epifaunal assemblages has been limited mainly due to the sampling challenges. It is known that each new area of seep has its own characteristic in terms of faunal structure, diversity, and chemical conditions, which makes them somewhat similar to others, but also makes them unique.

Chapter 2

This work represented the first meio-epifaunal characterization of the higher rank taxa associated with loose carbonate rocks in deep-water cold seeps, and provides insights into the initial (months) steps of colonization on denuded rocks. Differences were found between the distinct moments of colonization.

A total of 27 meio-epifaunal taxa formed the assemblage of natural rocks, many more than common deep-sea biogenic substrates. In this natural rock nematodes accounted for the highest abundances, followed by copepods. According seepage activities, inactive areas accounted for the highest mean number of individuals. Metazoan meio-epifauna discriminated at higher taxonomical ranks do not clearly cluster or segregate by one particular factor, location or activity-habitat. Different locations exhibit different hydrographic characteristics, with depth and temperature being the variables that best explain the biological scheme found.

After10.5 months of colonization on deployed substrates, the community was comprised of less meio-epifaunal taxa. In contrast to natural rocks, copepods accounted for the highest abundances, followed by nematodes. As in natural rocks, the highest mean density was found in inactive areas. Also, significant differences were found between habitats but not between seepage activities.

In our results, the highest densities of meio-epifauna were always associated with inactive sites. It was obtained that in average density Nematoda was dominant in rocks located in active and inactive sites, exceeding copepods in both cases. In experimental rocks, both in active and inactive sites, copepods were dominant. The seepage activity is factor that needs to be characterized in situ.

130 Meio-epifauna Costa Rica

The copepod dominance in the relatively short-term duration of the experiment suggest these mobile metazoans are pioneers during early stages of succession on hard substrates of the deep sea. Nematodes, in contrast, seem to dominate the developed meio-epifaunal communityof the natural carbonate rocks in Costa Rican cold seeps.

Contrary to previous studies of deep-sea hard bottom meio-epifauna, in which copepodes have been found to be the dominant component, the natural carbonate rocks studied here were dominated by nematodes. These results may suggest that community composition changes with the residence time of the substrate. A question remains open: How do nematodes colonize authigenic rock substrates in Costa Rica cold seeps?

We do not observe a clear pattern regarding the structural complexity of the habitat. Natural rock substrates located on sediments or in other types of habitats (with foundations species) did no showed clear patterns in mean number of taxa or densities. These relationships could be explored at lower taxonomic levels.

In the only OMZ site of this study (Quepos landslide location) the mean number of total taxa compared to the other locations, was the lowest. This suggests that oxygen deficiency may have an influence on the meiofaunal composition at higher taxonomic levels.

Chapter 3 In contrast to the high diversity of higher taxa/groups found associated with the rock substrates, in the woods only were found nine taxa. Copepods accounted for the highest abundances, followed by nauplii larvae, and nematodes.

Wood pieces in the deep sea provide additional hard substrate to be colonized by meio-epifaunal organism, increasing habitat complexity. However, given the short time frame of this study and the relative low degree of degradation of the Douglas fir wood by bacteria and the absence of wood borer bivalves, our experimental wood blocks represent only an early stage of colonization and faunal succession, possibly unrelated to the chemical and physical processes of degradation that take place in plant remains.

The lack of strong differences in higher taxa composition and density between seepage activity and between habitats within activities found in our study, may be attributed to the homogeneity of the wood blocks as substrate, the short duration of the experiment relative to wood degradation, but also to low sample size.

The dominance of the main group that structures meio-epifaunal communities (i.e., nematodes) could be limited due to a number of factors. These limiting factors include: Differences in chemical composition of the wood, time elapsed since sinking, food sources, and absence of wood-boring bivalves.

As wood substrates are expected to increase in frequency of occurrence in the coming years, due to extreme climate events which have increased the mobilization of such debris, our study gains relevance owing to the positive or negative consequences that this process may have on the colonization and dispersion of species. Chapter 6 131

Chapter 4

The present study is the first characterization of the copepod fauna associated with cold-seep experimental substrates in the deep sea of Costa Rica. Hard substrates placed on six different habitats provide a suitable habitat for epifaunal organism, favouring the presence of harpacticoid copepods.

It is highlighted that Copepoda was abundant and diverse at seeps hard substrates and are important early colonizers. The community structure, examined at the family level, differs significantly between the two principal substrates analyzed (authigenic rocks and wood blocks). A turnover in family dominance between Miraciidae and Ectinosomatidae, according to seepage activity, was observed.

In this study and all the samples analyzed, dominance of copepods was evident above the other taxa. This is remarkable if is considered that in most of the studies the second most abundant meiobenthic taxon after the numerically dominant Nematoda are copepods.

Benthic copepods may be playing an important role in the surveyed ecosystem, representing an important linkn in the cold seep food web in Costa Rica. It is known that in the upper food web they serve as food for many invertebrates and juvenile fishes, and in the lower portion of the web they feed to a large extent on the microbial flora and are therefore important in the recycling of benthic energy.

Chapter 5

Free-living nematodes are an abundant and diverse component of the meio-epifauna inhabiting natural authigenic rocks of Mounds 11 and 12 of Costa Rica. Nematodes because of their abundance and dominance in various substrates of groundwater play a key role in the maintenance of these communities that colonize extreme environments. According to some authors, the reasons why nematodes are successful in colonizing different environments are very poorly understood. ematode fauna was represented by 27 genera identified. The most abundant families were Xyalidae, Comesomatidae, and Linhomoeidae. Daptonema and Metalinhomoeus were the taxa accumulated the highest density in all survey. The results demonstrate the existence of slight differences between habitats regarding nematode assemblage structure. On the substrates, the assemblages were composed of nematode taxa with different trophic features. Non-selective deposit-feeders were dominant, followed by epigrowth feeders. In the Pacific region of Costa Rica, common families and genera to other regions of the world were found. However, its differentiation from other basins remains to be resolved at a specific level.

Due to their obvious importance, nematodes must thus be included in calculations of community trophic structure and community energy budgets of Pacific cold seep areas.

Although no work was done at the species level, it is known that some species of nematodes are considered to have a wide distribution as a result of trans-oceanic dispersal and the cosmopolitan distribution of a range of meiofauna taxa is probably the result of a variety of dispersal mechanisms (including resuspension and transport with currents), so it is expected to find new species for science in these environments, but also known species that inhabit other depths and habitats of the ocean. 132 Meio-epifauna Costa Rica

6.2 Recomendations

Because this experiment was conducted only at one early time stage (10.5 months), it will be necessary to study the temporal dynamics of susbtrates colonization and community assembly through longer chronological spans, to better understand micro to macrobiological scale processes associated with succession in these reducing environments.

Future colonization studies in the area should involve the use of several types of susbtrata, especially different kind of wood. The latter to confirm the settlement patterns found, and know their progression, especially to identify the role of copepods as early colonizer and their relation to wood degradation processes.

To better understand the colonization processes of meio-epifauna, it would be important to understand what happens at the first level of colonization. The meio-epifauna is in the second level, but the first is still uncertain.

It would be very useful to be able to measure in situ the dynamics of sulfide enrichment on on the wood surface, as well as other variables (e.g., pH, O2) to establish their influence.

It is needed to advance in taxonomic, biotic and ecological aspects of nematods and copepods community that dominate these substrates. It is critical to understand their role and strong relationships that can occur with other fauna. Taxonomically, nematode and copepod fauna of the Pacific of Costa Rica is poorly known, thus to go forward in the knowledge of the species and determine whether there are changes in the community at a lower taxonomic resolution.

Another focus for future studies can be related to the naupliar development stage, given the high number of specimens found both in natural and experimental substrates. That can help to elucidate on the role being played of these substrates in the development of these organisms.

Offshore Costa Rica and southern Nicaragua, Sahling et al., (2008) identified more than 100 seeps localities (on average one seep every 4 km); more recently Kluesner et al., (2013) documented 161 sites of potential fluid seepage only on the shelf and slope regions offshore Costa Rica. From the above, it is considered that this is a geomorphologically diverse area, that include the presence of various types of reducing environments. It will provide many points of future exploration and biology experimentation, which will allow advancing the knowledge of their communities in the Pacific coast.

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