Highly Seasonal Reproduction in Desmophyllum Dianthus from the Northern Patagonian Fjords Keri Feehan University of Maine, [email protected]

Highly Seasonal Reproduction in Desmophyllum Dianthus from the Northern Patagonian Fjords Keri Feehan University of Maine, Keriafeehan@Gmail.Com

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Spring 3-10-2016 Highly Seasonal Reproduction in Desmophyllum dianthus from the Northern Patagonian Fjords Keri Feehan University of Maine, [email protected]

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This Open-Access Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of DigitalCommons@UMaine. HIGHLY SEASONAL REPRODUCTION IN DESMOPHYLLUM DIANTHUS FROM THE

NORTHERN PATAGONIAN FJORDS

Keri A. Feehan

B.S. University of Maine, 2013

A THESIS

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

(in Marine Biology)

The Graduate School

The University of Maine

May 2016

Advisory Committee:

Rhian G. Waller, Professor of Marine Sciences, Advisor

Kevin Eckelbarger, Professor of Marine Sciences

Bob Steneck, Professor of Marine Sciences and Oceanography

THESIS ACCEPTANCE STATEMENT

On behalf of the graduate Committee for Keri A. Feehan I affirm that this manuscript is the final and accepted thesis. Signatures of all committee members are on file with the Graduate School at the University of Maine, 42 Stodder Hall, Orono, Maine.

Dr. Rhian G. Waller, Associate Professor March 2nd, 2016

iii

LIBRARY RIGHTS STATEMENT

In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of Maine, I agree that the Library shall make it freely available for inspection. I further agree that permission for “fair use” copying of this thesis for scholarly purposes may be granted by the Librarian. It is understood that any copy or publication of this thesis for financial gain shall not be allowed without my written permission.

Signature:

Date:

HIGHLY SEASONAL REPRODUCTION IN DESMOPHYLLUM DIANTHUS FROM THE

NORTHERN PATAGONIAN FJORDS

By: Keri A. Feehan

Thesis Advisor: Dr. Rhian G. Waller

An Abstract of the Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master of Science (in Marine Biology) May 2016

The purpose of this study was to determine the basic reproductive biology and seasonality of the Patagonian fjord coral Desmophyllum dianthus. Desmophyllum dianthus is a deep-sea solitary scleractinian found throughout the world’s oceans and an important benthic habitat builder. The Chilean Patagonian fjords are the only known location where this species occurs >50 m and thus are the only place to collect samples efficiently, effectively and economically. Corals were collected via SCUBA approximately every three months (when conditions permitted) from August 2012 to

September 2013 from three sites within the Northern Patagonian fjords – Lilihuape

(n=76) and Punta Huinay (n=59) in the Comau fjord, and Punta Mamurro (n=44) in

Reñihue fjord. The objectives of this study were to determine sexuality, reproductive mode, oocyte size, fecundity and seasonality using histological techniques.

Environmental data (temperature, salinity and light) was also analyzed to compare to seasonal reproductive trends and bring reproductive data into context. This study

determined that Desmophyllum dianthus is dioecious (gonochoristic), having both male and female individuals. This species is also highly seasonal, spawning in the austral winter (August) and beginning gamete production in early spring. The fjord was coolest and most saline in August 2012, potentially cueing spawning. No planula larvae were found in any of the 8,000 histological sections. Due to the presence of late stage oocytes in August 2012, it is likely D. dianthus’s mode of reproduction is spawning rather than brooding. However this distinction could not be determined in this study. Oogenesis starts in September producing previtellogenic oocytes (size range: 25 – 200 μm) that slowly developed into vitellogenic oocytes by June.

Vitellogenic oocytes ranged from 200 – 380 μm. Fecundity is relatively high compared to other deep-sea scleractinians, ranging from 2,448 (± 5.13 SE) to 172,328 (±103.67

SE) average potential oocytes per polyp. This research provides the first insight into

Desmophyllum dianthus reproductive biology and yields an important baseline for continuing work on this benthic habitat builder in the Chilean fjords, and in the deep ocean.

ACKNOWLEDGEMENTS

This research would not have been possible without the collaboration between the

University of Maine and the Punta Huinay Scientific Field Station. I would like to thank National Geographic and the National Science Foundation for the finical support for this project. A tremendous thank you to Rhian G. Waller, Laura Grange,

Chris Riguad, Vreni Haussermann, Gunter Forsterra and all divers & scientists at the

Punta Huinay scientific field station who collected the samples for this study. I would also like to thank undergraduates M. Halfman, A. Rossin, and E. Hartill for their help with decalcifications and staining. I would like to thank Damian Brady for over a 9,000 data points worth of salinity conversions. I am deeply grateful to my friends, family and fiancé Travis Conley for their support and love over the last two years. To my committee, Kevin Ecklebarger and Bob Steneck it has been a joy to know you and learn from you over the last several years, thank you for the all the great conversations and inspiration. To my advisor and mentor Rhian Waller, thank you for cultivating my passion for marine biology, thank you for every opportunity you have put my way, and thank you for your patience, kindness and forthright honesty. It is a pleasure to know you; you have helped me learn but most importantly you have helped me grow into the scientist I am today. I will be forever grateful for you and all who I have meet on my journey at the University of Maine.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS…………………………………………………………………………………iv

LIST OF TABLES………………………………………………………………………………………….…vii

LIST OF FIGURES……………………………………………………………………………………….…viii

CHAPTER

1. INTRODUCTION……………………………………………………….………………………………….1

2. HYPOTHESIS……………………………………………………………………….……………………….8

3. MATERIALS AND METHODS……………………………………………………………...... 9

4. RESULTS……………………………………………………………………………...….…………………14

4.1. Environmental Analysis…………………………………………………………….…………14

4.1.1. Lilihuape………………………………………………………………………………...... 14

4.1.2. Punta Huinay………………………………………………………….………………..….14

4.1.3. Punta Mamurro……………………………………………………………………….…..15

4.2. Gametogenesis…………….………………………………………………………………….….18

4.3. Fecundity…………………………………………………………………………………….………22

4.4. Size and fecundity………………………………………………………………………….……23

5. DISCUSSION……………………………………………………………………….…....…….……...... 27

REFERENCES………………………………………………………………………………..…………….34

APPENDIX A. SUMMARY OF REPRODUCTIVE DATA BY SITE………….…….……..40

APPENDIX B. SUMMARY OF FECUNDITY DATA……………………………....………….47

APPENDIX C. SUMMARY OF COLD-WATER SCLERACTINIAN

REPRODUCTIVE DATA………….…………………………………………………………………….48

BIOGRAPHY OF THE AUTHOR…………………………………………………….….…………..50

LIST OF TABLES

Table A.1. Summary of size parameters and sex of Desmophyllum

dianthus individuals collected at Lilihuape in the Comau fjord….…………..….40

Table A.2. Summary of size parameters and sex of Desmophyllum

dianthus individuals collected at Punta Huinay in the Comau fjord.…….…….43

Table A.3. Summary of size parameters and sex of Desmophyllum

dianthus individuals collected at Punta Mammuro in the Renihue fjord..…..45

Table A.4. Summary of size parameters and sex of Desmophyllum

dianthus individuals collected at Cabudahue in the Renihue fjord……………..46

Table B.1. Summary of fecundity (opp), average oocyte diameter,

polyp area and number of reproductive mesenteries in females

from all locations……………………………………………………………………………………….47

Table C.1. Summary of sexuality, mode of reproduction and

maximum oocytes sizes in all deep-sea Scleractinians published

to date………………………………………………………………………………………………………48

vii

LIST OF FIGURES

Figure 1. Map of study sites in the Comua and Reñihue fjords,

Southern Chile ……………………………………………………………………………………..…..10

Figure 2. Height (H), length (L) and width (W) measurement

specifications for Desmophyllum dianthus..……………………………..…………..…..12

Figure 3. Histological sections of Desmophyllum dianthus

reproductive anatomy………………………………………………….…………………………...13

Figure 4. Seasonality at Lilihuape in terms of environmental and

reproductive data from August 2012 to September 2013….……………………...16

Figure 5. Seasonality at Punta Huniay in terms of environmental

and reproductive data from August 2012 to September 2013……………….....17

Figure 6. Seasonality at Punta Mamurro in terms of environmental

and reproductive data from August 2012 to September 2013…………………..18

Figure 7. Percent frequency of oocyte diameter per month

from females collected in the Comau fjord (Lilihuape and

Punta Huinay collection sites)……………………………………………………………..…....21

viii

Figure 8. Percent frequency of oocyte diameter per month from females collected in the Comau fjord (Lilihuape and

Punta Huinay collection sites)……..….…………………………………………………..…...22

Figure 9. Average potential fecundity by month at Lilihuape..…………………..24

Figure 10. Average potential fecundity in the Comau fjord in June

2013………………………………………………………………………………………………………....24

Figure 11. Size of collected Desmophyllum dianthus individuals by location.………………………………………………………………………………………………..…..25

Figure 12. Average potential fecundity as a function of polyp area (length*width) from female corals (N = 7) collected from

Lilihaupe in February 2013……………………………………………………………………...…25

Figure 13. Average fecundity as a function of polyp area

(length*width) of female corals (N = 3) collected from Lilihaupe

in June 2013.……………………….……………………………………………………………….…..26

Figure 14. Average Potential fecundity (opp) in the Renihue fjord (Punta Mammarro) in March 2013 as a function of polyp area (N = 3)…….……………………………………………………………………….....…..26

CHAPTER ONE

INTRODUCTION

The Patagonian region is located in South America, starting at approximately

37° south, extending all the way to the tip of Cape Horn and spanning east to west across Chile and Argentina. Chile has one of the longest coastlines in the world making the ocean and its resources important to the Chilean people for food, income and recreation. Furthermore, Southern Chile encompasses one of the most extensive fjord systems in the world (Acha et al., 2004; Iriarte et al., 2010). The Patagonian fjord region resulted from glacial erosion and subsequent retreat of the ice caps after the last glacial maximum (Borgel, 1970; Pantoja et al., 2011). Approximately 15,000 yr. ago the Patagonian ice sheet retreated forming the Northern and Southern Patagonian ice fields that are found in Chile today and exposed 84,000 km of coastline composed of fjords, islands and channels (Häussermann & Förstera 2009; Silva & Vargas 2014).

Fjords are among the most biogeochemically dynamic places on the earth (Gattuso et al., 1998). The biota in this region are heavily influenced by physical and environmental factors such as the Pacific Ocean, Southern Ocean and relating currents, the Andes

Mountains, volcanic activity and glaciers.

Multiple ocean currents influence the fjord system. The major currents are the

Peru Counter Current (PC), the Humboldt Current (HC), and the Antarctic Circumpolar

Current (ACC) (Silva et al., 2009). The Peru-Chile undercurrent (PCU) and Cape Horn

Current (CH) are minor ocean currents off of southern Chile. All five currents are composed of cold-water bodies, providing higher than average oxygen content to this

1 system (Silva & Vargas 2014). In a study by Silva & Vargas (2014), of the 90 fjords studied in Southern Chile, 86 were oxic (> 2 mL L-1) and only 4 were hypoxic (< 2 mL L-

1). None was found to be anoxic (0 mL L-1). All of these ocean currents play an important role in Patagonian oceanography; however the most influential water mass in the fjords is Sub-Antarctic Water (SAAW) (Häussermann & Förstera 2009). This cold, oxygen rich water is upwelled due to a strong thermohaline circulation.

The average annual rainfall in the Patagonian region is 1705 mm yr-1

(Häussermann & Förstera 2009). This massive input of fresh water combined with large volumes of river/glacial runoff creates a thick freshwater layer (10 m) and drives the thermohaline circulation. The robust thermohaline circulation subsequently upwells

SAAW, mixing with the fresh water and resulting in the formation of Modified Sub-

Antarctic Water (Häussermann & Förstera 2009).

High terrestrial runoff from surrounding forests provides nutrients and tannins to the system (Pantoja et al., 2011). As tannins severely limit or completely obstruct light attenuation, waters below the thick freshwater layer are almost completely dark.

In addition, volcanic and glacial activity in the region regularly delivers micronutrients, such as iron, to the fjords (Iriarte et al. 2014). All of these complex interacting physical and environmental factors make the Patagonian fjords dynamic, and structures the biodiversity and biogeography found in the region.

This combination of high oxygen content, terrestrial runoff, volcanic activity and multiple currents that provide larval transport have created a biodiversity hot spot within the Patagonian fjords (Häussermann & Förstera 2009; Pantoja et al. 2011). Not

2 only are the fjords rich with a diversity of species, but they are also home to a rare phenomenon known as deep-water emergence. This phenomenon has only been documented at four other places in the world - Alaska (Stone & Shotwell 2007; Waller et al., 2014), New Zealand (Parker et al., 1997), Norway (Brooke & Järnegren 2013), and

Sweden (Wisshak et al., 2005). Deep-water emergence occurs when a deep-sea species inhabits depths shallower than their usual distribution due to bathyal- or abyssal-like conditions. A primary example of a deep-water emergent species in the Patagonian fjords is the cold-water coral Desmophyllum dianthus (Esper, 1794).

Cold-water corals are a diverse group of Anthozoa usually inhabiting continental shelves, seamounts and deep-sea ridges (Roberts, 2009). All cold-water corals are azooxanthellates, meaning they do not harbor photosynthetic algal symbionts, but feed on zooplankton and detritus. Since cold-water corals do not need sunlight to obtain energy this allows them to live typically between 200 and 6,000 m

(Keller, 1976; Freiwald et al. 2004), though many species live outside these boundaries (Cairns, 2007, Waller et al., 2014). Cold-water corals can be important ecosystem engineers that provide habitats to a variety of species including invertebrates and commercially important fishes and crustaceans (Roberts &

Hirshfield 2004; Roberts et al., 2006; Brancato et al., 2007; Stone & Shotwell 2007;

Braga-Henriques et al., 2013). Despite their diversity and their important role in benthic deep-sea communities, the biology and ecology of cold-water corals is not well understood, especially in comparison to their shallow-water counterparts

(Roberts, 2009). This is primarily because of the logistical difficulties in studying deep-

3 sea species. Collections are difficult and expensive, and often only small samples sizes can be taken. Furthermore, there is almost no guarantee of sampling in the same place twice, making ecological studies sparse.

Obviously reproduction is a fundamental ecological process, vital to the propagation and success of a species over time. Of the 3,000+ known species of cold-water corals, information on reproductive biology is known for fewer than 50 species (Feehan & Waller 2015). Reproduction can also be a sensitive indicator of environmental stress (Van Veghel & Bak, 1994; Richmond, 1997; Zakai et al., 2000;

Harrison & Ward, 2001; Bongiorni et al., 2003; Waller & Tyler, 2005). Reproduction and somatic growth are two key life processes that utilize energy within an organism. When stresses occur, such as disease, temperature change, loss/reduction of food source or low oxygen content, energy is diverted away from one or both of these areas to compensate for the stress. This relationship has been demonstrated numerous species, across multiple phyla, from echinoderms to fishes

(Pankhurst &Van Der Kraak, 1997; Lawrence & Herrera, 2000). Prior to this study the reproductive mode and seasonality of Desmophyllum dianthus was unknown.

Desmophyllum dianthus is a cosmopolitan, solitary, deep-water scleractinian

(Förstera & Häussermann, 2003; Addamo et al., 2012). Desmophyllum dianthus is a slow growing (0.5-2.3 mm a year) and long-lived (~200 yr) species (Addamo et al.,

2012), and usually lives on steep, rocky ledges or overhangs between 1,000 and 2,500 m. In the Patagonian fjords, however, D. dianthus is found in high densities (1,500 individuals m-2) at depths as shallow as 8 m (Förstera & Häussermann, 2003).

4 Despite the dynamic and diverse ecosystem found in the Patagonian fjords, it is one of the least studied marine environments in the world (Häussermann & Förstera

2009). Only minimal oceanographic and biological frameworks have been established, and there are currently no marine management plans in place. Within the past two decades anthropogenic impacts such as logging and fish aquaculture have significantly increased within this region. The Patagonian region is a highly productive, diverse and dynamic ecosystem and may be changing before it can be understood in its natural state (Häussermann & Förstera 2009).

A major concern, with respect to anthropogenic impacts, is the effect of fish aquaculture within the fjords. Fish aquaculture was introduced in 1980 and has significantly increased in the past ten years (Buschmann et al., 2009; Niklitschek et al.,

2013). Salmon farming contributes the highest percentage of aquaculture within the

Patagonian fjords, and Chile is the second highest producer of farmed salmon in the world (Niklitschek et al., 2013). Impacts of aquaculture have been extensively studied in the Norwegian fjords, and can cause eutrophication, increased frequency of harmful algal blooms, and hypoxia or anoxia to which fjords are already particularly prone (Wu,

1995; Skogen et al., 2009). Fjords are often subject to hypoxia because of a sill at the fjord entrance. The sill prevents the deep dense water from mixing thus oxygen is not replenished after aerobic processes leading to oxygen depletion. Fish aquaculture has been demonstrated to significantly decrease species diversity within a 550 m radius of pens and cages—translating to a significant loss of diversity for a 0.95 km2 area (Kutti et al., 2007). These undesirable impacts can stress organisms, especially sessile benthic

5 organisms. In Chile, salmon farmers have to pump pure oxygen into the salmon pens because they are so densely packed that the water would be anoxic without the enchantment (Försterra et al., 2014). Chile does not have an effective or up to date marine management plan in place to properly regulate fish aquaculture (Buschmann et al., 2009). It is therefore important to understand how extensive the impact of salmon aquaculture is on the diversity of benthic communities within the Patagonian fjords.

The Patagonian fjords are one of the world’s largest fjord systems, with high biodiversity, high productivity, and home to a deep-water emergent species. Ensuring the sustainable management of these Chilean fjords and its resources will require detailed biological information on both the benthic and pelagic species present within this system.

Deep-water emergence within the fjord also allows a unique opportunity to study ecological timeseries on a deep-sea species. Desmophyllum dianthus is usually found at over 1,000 m depth, but is found at just 10m in the Patagonian fjords

(Häussermann & Förstera 2009), depths that are accessible by SCUBA. This allows repetitive sampling from a single population and thus the ability to conduct ecological studies on a, normally, deep-sea species. These shallow coral populations are windows into the abyss, providing a living laboratory in which to do in situ studies. An ecological study to understand the biology, reproduction and seasonality of D. dianthus is not only important for increasing our understanding cold-water coral ecology in general, but also important in understanding a major benthic species found throughout the Patagonian fjord region, and the global deep ocean. The Patagonian fjords are the only known

6 location for easy access to D. dianthus populations. Investigating D. dianthus reproduction may also shed light on important environmental impacts.

The purpose of this study was to investigate the reproductive biology and seasonality of the cold-water coral Desmophyllum dianthus in the Northern fjords of

Patagonia. The objectives of this study were to determine the sexuality, reproductive mode, oocyte size, fecundity and seasonality of D. dianthus using histological techniques. Reproductive output (fecundity) will be compared between sites.

7 CHAPTER TWO

HYPOTHESIS

This study will address five research questions with the relating hypotheses:

1. Is D. dianthus gonochoristic or hermaphroditic?

Hypothesis: Individuals have only single sex (male or female) gametes present in mesenteries.

2. What reproductive mode does D. dianthus utilize?

Hypothesis: Desmophyllum dianthus uses broadcast spawning as its primary mode of reproduction.

3. Does D. dianthus reproduce seasonally, continuously or semi- continuously?

Hypothesis: There is a significant difference in reproductive parameters between months.

4. Does fecundity relate to polyp height, length or width?

Hypothesis (a): There is a significant relationship between fecundity and polyp height.

(b) There is a significant relationship between fecundity and polyp length. (c) There is a significant relationship between fecundity and polyp width.

5. Do the reproductive parameters differ between collection sites?

Hypothesis: There is a significant difference in reproductive parameters between sites.

8 CHAPTER THREE

MATERIALS AND METHODS

Corals were collected via SCUBA approximately every three months from

August 2012 to September 2013 from three sites within the northern Patagonian fjords – Lilihuape (n = 76) and Punta Huinay (n = 59) in the Comau Fjord, and Punta

Mamurro (n = 44) in Reñihue Fjord (Fig. 1.1). However due to the remote location of

Punta Mamurro samples were only collected three times throughout the 13 month study. Hobo temperature and light loggers were deployed at all three sites to collect temperature, salinity and light (illuminance) data throughout the entire study. The

Huinay Scientific Field Station was the base camp for the field portion of this study. All samples were fixed in 10% formalin with a borax buffer and transported to the Ira C.

Darling Marine Center at the University of Maine for further analysis.

Upon arriving at the Darling Marine Center each sample was assigned a random identification number to prevent the biasing of the results. The height, length and width of all individuals were measured prior to histological analysis (Fig. 1.2). Each coral was decalcified in Rapid Bone Decalcifer to remove their skeletons, before histological processing. After decalcification corals were placed in Bouins solution to complete decalcification, improve staining and firm tissue. Approximately 5 times the volume of the polyp tissue of Bouins solution was added to each individual coral falcon tube, varying from 15 mL to 45 mL.

Figure 1.1: Map of study sites in the Comua and Reñihue fjords, Southern Chile. Black dots indicate the three collection sites Lilihuape (L), Punta Huinay (PH) and Punta Mamurro (PM). The Huinay Scientific Field Station is represented by an asterisk (*). Desmophyllum dianthus individuals were collected at these sites between August 2012 and September 2013.

Figure 1.2: Height (H), length (L) and width (W) measurement specifications for Desmophyllum dianthus.

After 36 hours in Bouins solution all corals were transferred to 30% ethanol solution. Coral were then serially dehydrated by 10% ethanol increments for 30 minutes. At 70% ethanol three to five mesenteries were dissected out of each polyp and put into a labeled histology basket. Batches of 15 or 20 baskets were then serially dehydrated from 70 – 100% ethanol by 10% ethanol increments (with the exception of including 95% ethanol between the 90% and 100%) for 30 minutes. Baskets were left in

100% ethanol over night (~10 hrs). The following morning baskets were cleared in three

30 min submersions of toluene. After the final toluene submersion baskets were

11 transferred into melted paraffin wax (Paraplast Plus, Fisher Scientific) within an oven set at 60 °C. Baskets went through three wax changes over a 48 hour time period.

Dissected coral mesenteries were then embedded into paraffin wax blocks. All blocks were sectioned with a Leica Microtome leavening 45 μm between sections

(width of nucleus) and slicing 5 μm sections that were transferred to a water bath set at 45 °C. Sections were then mounted on glass slides and left to dry on slide warmers for ~8 hrs. Histological slides were then stained in Mason’s Trichrome, cover slipped and left to dry for one day before viewing.

Histological slides were examined using an Olympus (CX31) compound microscope with a Motic video camera attachment. Male and female individuals were identified, and female oocytes and male spermatocysts were staged. Oocytes were sorted into two stages: vitellogenic and previtellogenic (Fig. 1.3). Spermatocysts were categorized into four stages of maturity, I through IV (increasing in maturity) (Fig. 1.3). One hundred spermatocysts were counted and staged for each male individual, spermatocyst frequency diagrams were constructed from these results. All images were captured using Motic Image Plus and analyzed using ImageJ software (NIH) to calculate oocyte diameter (a “Feret” diameter function was used to determine the area of the oocyte as if it were a perfect circle). One hundred random oocytes were measured using this method and oocyte size-frequency diagrams were constructed from results.

Total fecundity was calculated by counting all previtellogenic and vitellogenic oocytes (that have a nucleus present) within the three mesenteries. For the purpose of

12 this study we are defining potential fecundity as an individual’s maximum potential reproductive output (# of oocytes) based on the number of reproductive mesenteries present. To determine fecundity the total number of oocytes was quantified in three mesenteries and then averaged, yielding a mean fecundity per mesentery. To calculate potential fecundity the number of reproductive mesenteries was determined by dissecting out 12 – 40 mesenteries

Figure 1.3: Histological sections of Desmophyllum dianthus reproductive anatomy. (a) Female showing previtellogenic oocytes (pv). (b) Female showing vitellogenic oocyte (V). (c) Late stage oocytes (lso). (d-f) Spermatogenesis: males showing spermatocyst stages, from least mature (stage I) to mature (stage IV).

13 per location and quantifying how many were reproductive. The ratio of total mesenteries to reproductive mesenteries was extrapolated to reflect total reproductive mesenteries within the 96 present within Desmophyllum dianthus.

Seasonality was determined by a nonparametric Each Pairs Wilcoxon test comparing average oocyte size frequencies and average fecundities between months in females, and stages of spermatocyst growth in males throughout the study period.

Linear regression determined relationships between coral height, length, width and fecundity. An additional nonparametric Each Pairs Wilcoxon test was used to compare sizes (height, length and width) between locations. Environmental data (temperature, salinity and light (illuminance) was also be analyzed to compare to seasonal reproductive trends to bring reproductive data into context.

14 CHAPTER FOUR

RESULTS

4.1 Environmental Analysis

4.1.1. Lilihuape

Out of the 14-month environmental observation period the highest monthly temperature average was observed in February 2013 (13.05 °C) (Fig. 1.4). The lowest monthly temperature average was in August 2012 (9.79 °C). The general trend was warming from September to April and cooling from May to August. Salinity was lowest in January 2013 (26.1 PSU) and highest in August 2012 (28.17 PSU) (Fig. 1.4).

The light intensity logger only recorded from August 2012 to July 2013 due to sensor failure post July. During that 12 month period light intensity was greatest in

November 2012 (1.37 lx) and lowest in June 2013 (0.03 lx) (Fig. 1.4). Light, temperature and salinity loggers were deployed at 19.5 m at Lilihuape.

4.1.2. Punta Huinay

Out of the 14-month environmental observation period the highest average monthly temperature was observed in February 2013 (12.34 °C) (Fig. 1.5). The lowest average monthly temperature was in August 2012 (9.96 °C). The general trend was warming from September to February and cooling from March to August. Salinity was lowest in June 2013 (25.73 PSU) and highest in August 2012 (31.50 PSU) (Fig. 1.5).

Light intensity (illuminance) was greatest in February 2013 (0.28 lx). October 2012, and April through August 2013 had an average of 0 lx (Fig. 1.5). It is important to note

15 that a large sea star had to be removed from on top of the light sensor three separate times, which undoubtedly affect light intensity readings. Light, temperature and salinity loggers were deployed at 25 m at Punta Huinay.

Figure 1.4: Seasonality at Lilihuape in terms of environmental and reproductive data from August 2012 to September 2013. (a) Salinity and temperature N = 9749. (b) Average light intensity per month from August 2012 to July 2013. (c) Average Oocyte diameter per month: August N = 2; November 2012 N = 1; February 2013 N = 7; June 2013 N = 5; September 2013 N = 1. (d) Percent frequency of spermatocyst stages per month. August N = 1; February N = 4; June N = 8. Error bars indicate standard error.

Figure 1.5: Seasonality at Punta Huniay in terms of environmental and reproductive data from August 2012 to September 2013. (a) Salinity and temperature N = 9605. (b) Average light intensity per month from August 2012 to September 2013. (c) Average Oocyte diameter per month: April 2013 N = 1 and June 2013 N = 2. (d) Percent frequency of spermatocyst stages per month. April 2013 N = 6; June 2013 N = 3; September 2013 N = 1. Error bars indicate standard error.

4.1.3. Punta Mamurro

Out of the 14-month environmental observation period the highest average monthly temperature was observed in February 2013 (12.35 °C) (Fig. 1.6). The lowest average monthly temperature was in August 2012 (9.31 °C). The general trend was warming from September to February and cooling from March to August. Salinity was

17 lowest in March 2013 (26.62 PSU) and highest in August 2012 (28.23 PSU) (Fig. 1.6).

Light intensity was greatest in November 2012 (0.72 lx) and lowest in May and June

2013 with a monthly average of 0 lx (Fig. 1.6). Light, temperature and salinity loggers were deployed at 27 m at Punta Mamurro.

Figure 1.6: Seasonality at Punta Mamurro in terms of environmental and reproductive data from August 2012 to September 2013. (a) Salinity and temperature N = 9667. (b) Average light intensity per month from August 2012 to September 2013. (c) Average Oocyte diameter per month: March 2013 N = 2. (d) Percent frequency of spermatocyst stages per month. August 2012 N = 5 and March 2013 N = 7. Error bars indicate standard error.

18 4.2. Gametogensis Desmophyllum dianthus is a gonochoric species, having distinctly male and female individuals. However this is not apparent on outward appearance, only by dissection or histological section can male or female individuals be identified. No hermaphrodites were found in this study. Out of 195 corals collected 22 were females and 36 were males. Desmophyllum dianthus has a sex ratio of approximately 2:1 males to females. Oocytes and spermatocysts were found embedded in the mesentery. Oogonia and spermatogonia originate from the mesenterial filament. Stage one spermatocysts were small (average diameter:

108.79 μm ± 5.38 SE) usually standing alone surrounded by the mesenterial filament with patchy spermatids within (Fig. 1.3d). Stage two spermatocysts have a distinct lumen with a thick border of densely packed developing sperm and were 234.89 μm

(± 20.61 SE) in diameter (Fig. 1.3e). Stage three spermatocysts also have a thick boarder of densely packed spermatids but the lumen is now being filled in with lightly or densely packed sperm. Stage three spermatocyts were 267.93 μm (± 18.58

SE) in diameter (Fig. 1.3f). Both Stage two and three spermatocysts were usually closely packed together next to other spermatocysts. Stage four spermaocysts were large (average diameter: 271.07 μm ± 13.26 SE) with dense mature sperm and visible sperm tails (Fig. 1.3f).

Previtellogenic oocytes range from 25 – 200 μm in diameter while vitellogenic oocytes ranged from 200 – 380 μm (Fig. 1.3). Two female’s oocytes were undergoing meiosis when collected in August 2012 (Fig. 1.3). In the Comau fjord females had high frequencies of previtellogenic oocytes in November, February, and

19 April 2012 as well as September 2013 (Fig. 1.7 and 1.8), In June 2013 the Comau females had approximately equal frequencies of previtellogenic and vitellogenic oocytes. In August 2012 all females had only vitellogenic oocytes present within the mesenteries (Fig. 1.8). Females collected in September 2013 had the smallest average oocyte diameter (50.96 μm) while August 2012 females had the greatest oocyte diameter (301.25 μm) (Fig. 1.8). The general trend for females in the Comau fjord was small previtellogenic oocytes starting development in September slowly growing in size and yolk content until they transitioned to mostly vitellogenic oocytes in June. Oocytes developed uniformly and steadily, at no point in this study were small previtellogenic oocytes and large vitellogenic oocytes (as seen in corals with continuous reproduction) in a single sample. Mean oocyte diameters per month at the Lilihuape location were highly statically significant (P-value <0.0001 for all comparisons with the exception of September 2013 Vs. November 2013 having a

P-value: 0.0003) confirming that D. dianthus is highly seasonal. In the Reñihue fjord, female samples were only collected in March 2013 thus no overall trend can be reported. However, average oocyte diameter of Renihue females in March follows the general seasonal trend seen in the Comau fjord (Fig. 1.4, 1.5, and 1.6).

Figure 1.7: Percent frequency of oocyte diameter per month from females collected in the Comau fjord (Lilihuape and Punta Huinay collection sites). August 2012 N = 2; November 2012 N = 1; February 2013 N = 7; April 2013 N = 1; June 2013 N = 5; September 2013 N = 1.

Figure 1.8: Percent frequency of oocyte diameter per month from females collected in the Comau fjord (Lilihuape and Punta Huinay collection sites). Error bars represent standard error. Black arrows indicate median oocyte diameter (μm)

21 4.3. Fecundity

Females from Lilihuape in February 2013 had the highest average potential fecundity at 172,328 (±103.67 SE) oocytes per polyp (opp). The lowest average potential fecundity was in August 2012 at Cabudahue in the Reñihue fjord: 2,448 (±

5.13 SE) Over this study’s observational period no definitive seasonal fecundity trend could be determined at any of the locations. However, Lilihuape had the most complete data set and fecundity seems to peak in February (Fig. 1.9). The average fecundity at Lilihuape in June 2013 is nearly ten times greater than the average fecundity at Punta Huniay that same month (Fig. 1.10). Individual mesenteries generally had the same number of oocytes. Number of oocytes (fecundity) varied greatly from individual to individual but generally oocyte counts were within a

Standard Error of ±71.6 between mesenteries. The greatest variance in oocytes between mesenteries (standard error of ±204.4) was seen was in one individual with

1300 oocytes in one mesentery and approximately 700 oocytes in the other two mesenteries.

4.4. Size and Fecundity

There was a significant difference in size in terms of polyp height, length and width between the four collection locations (Fig. 1.11). There is a significant difference in height, length and with between Lilihuape and Punta Huniay (height: p- value = 0.0001, length p-value = 0.0001 and width p-value = 0.0001). There was no significant difference in terms of height, length or width between Lilihuape and

22 Punta Mamurro. However a significant difference was found in term of height, length and width between Punta Mamurro and Punta Huniay (height: p-value =

0.0001, length p-value = 0.0001 and width p-value = 0.0001). In general Punta

Huniay individuals were small while Lilihuape and Punta Mamurro individuals were large and thick. No significant trend was found between fecundity and polyp area at

Lilihaupe in February or June 2013 (R2 = 0.164418 and R2 = 0.29351 respectively)

(Fig. 1.12 and 1.13). Nor was a trend found between polyp area and fecundity at

Punta Mamurro in the Renihue fjord (Fig. 1.14). The size of first reproduction in females was: Height: 33 mm, Length: 13 mm, Width: 11.5 mm, and Area; 149.5 mm.

Size of first reproduction in males was: Height: 43.1 mm, Length 10.5 mm, Width 7.8 mm and Area: 81.9 mm.

Figure 1.9: Average potential fecundity by month at Lilihuape. Error Bars Indicate standard error. August 2012 N = 2; November 2012 N = 1; February 2013 N = 7; April 2013 N = 1; June 2013 N = 5; September 2013 N = 1.

Figure 1.10: Average potential fecundity in the Comau fjord in June 2013. Error bars indicate standard error. Lilihuape N = 3 and Punta Huinay N = 1.

Figure 1.11: Size of collected Desmophyllum dianthus individuals by location. Error bars indicate standard error. Cabudahhue N = 17; Punta Mamurro N = 44; Punta Huinay N = 55; Lilihuape N = 74.

Figure 1.12: Average potential fecundity as a function of polyp area (length*width) from female corals (N = 7) collected from Lilihaupe in February 2013.

Figure 1.13: Average fecundity as a function of polyp area (length*width) of female corals (N = 3) collected from Lilihaupe in June 2013.

Figure 1.14: Average Potential fecundity (opp) in the Renihue fjord (Punta Mammarro) in March 2013 as a function of polyp area (N = 3).

26 CHAPTER 5

DISCUSSION

The purposes of this study were to describe the sex, reproductive mode and seasonality of Desmophyllum dianthus. These aims were focused by five hypotheses: 1. Is D. dianthus gonochoristic or hermaphroditic? 2. What reproductive mode does D. dianthus utilize? 3. Does D. dianthus reproduce seasonally, continuously or semi- continuously? 4. Does fecundity relate to polyp height, length or width? 5. Do the reproductive parameters differ between collection sites? Each hypothesis will be addressed and discussed in order.

To answer the first hypothesis Desmophyllum dianthus is gonochoristic. This was evident after a total of 8,000 histological sections of 196 individuals, all either contained oocytes, sperm or were non-reproductive. None of the corals were observed to have both male and female gametes. Gonochorism is highly common in cold-water scleractinians (Waller, 2005; Brooke & Järnegren; 2013, Feehan & Waller,

2015), though the opposite is true in tropical shallow water scleractinians, where hermaphroditism is common (Baird et al., 2009). Gonochorism is postulated to be an ancestral trait in scleractinia (Harrison, 2011). With increasing reproductive studies on solitary cold-water scleractinian species, there is increasing support that cold-water azooxanthellate may have given rise to zooxanthellate corals (Veron, 1995).

The primary mode of reproduction in all scleractinians (zooxanthellates and azooxanthellates) is broadcast spawning (Harrison & Wallace, 1990). Many corals utilize a timed gamete release triggered by environmental cues (such as temperature,

27 lunar cycle, food availability, etc.) to increase fertilization success. Spawning events are commonly examined in shallow water species (Babcock et al., 1986; Richmond &

Hunter, 1990; Richmond, 1997; Mangubhai & Harrison, 2008), but significantly less is known about cold-water coral spawning events due to difficulties surrounding their observation and collection. Timing of food fall (particulate organic matter) has been speculated to trigger various aspects of deep-sea invertebrate reproduction (Tyler et al., 1992; Tyler et al., 1993; Eckelbarger & Watling. 1995; Gooday, 2002; Young, 2003;

Mercier & Hamel, 2009). Some cold-water corals reproduce seasonality and may also be cued by seasonal food fluxes to the benthos or other environmental factors

(Waller & Tyler, 2005; Mercier et al., 2011). Lunar rhythms have also been linked to reproduction in some species of deep-sea invertebrates, including a species of cold- water coral (Mercier et al., 2011). It is likely D. dianthus is a broadcast spawner, as no brooded larvae were observed in this study. Oocytes were observed from early oogenesis in September to late stage vitellogenesis in August. Germinal vesicle break down in late stage oocytes indicates fertilization in mid August 2012, although it is unknown whether fertilization took place inside or outside of the polyp. Again based on the absence of planula larvae in any of the histological sections and the start of oogenesis in September it is highly likely that D. dianthus used broadcast spawning as its mode of reproduction.

Desmophyllum dianthus is highly seasonal. Gametogenesis takes approximately 11 months: gametes form in September and germinal vesicle break down in late stage oocytes (and likely spawning) takes place the following August, at

28 the end of the Austral winter. Spermatogenesis also took 11 months in some cases although in others spermatocytes matured sooner and stayed mature until speculated release in August. This is not uncommon in invertebrates, as spermatogenesis is generally less energetically expensive than oogenesis.

Patagonia is located in a highly seasonal temperate environment with many seasonal cues – thus it is not surprising that Desmophyllum dianthus utilizes these cues for reproductive purposes. The most apparent external cues are temperature and salinity. At all locations the water was coolest and most saline in August 2012 and 2013. It is not possible to separate out which of these cues may trigger gamete release. It is very common for a shallow water scleractinian species to use temperature as a cue for gamete release. Waters begin warming in September, which may initiate gametogenesis. There is no apparent trend between light intensity and reproduction, though lunar phase may also trigger gamete release in D. dianthus.

Oocytes lacking a germinal vesicle, which breaks down just before fertilization

(Stricker et al., 2013), were observed on August 10 and 11th 2012, just 9-10 days after the full moon. In a study by Richmond and Hunter (1990) some scleractinian species in Guam spawned 7-10 days after the July full moon. A similar phenomenon may be happening in the Patagonian fjords with D. dianthus spawning after the August full moon. Another deep-sea scleractinian, Flabellum angulare, releases gametes

(through broadcast spawning) with the new moon (Mercier et al., 2011). Although D. dianthus seems to be ready to spawn in between the full and quarter moon, the

29 study by Mercier and others (2011) bring lunar rhythm as a viable cue for cold-water corals.

Individual mesenteries generally had the same number of oocytes; this number varied greatly from individual to individual but generally count were within a

Standard Error of ±71.6 (Variation in oocytes per mesentery: smallest: ±7.09 SE; greatest: ±204.4). In studies published to date, deep-sea scleractinian species maximum oocytes diameters range from 100 – 5,167 μm (Brooke & Tyler 2003 and

Waller et al., 2008 respectively) with an over all average maximum oocyte diameter of 1,000 μm (average of 28 species – See Appendix C). Desmophyllum dianthus maximum oocyte diameter (380 μm) is relatively small compared to other deep-sea scleractinians (Brook & Tyler 2003; Burgess & Babcock 2005; Waller, 2005, Waller et al., 2008; Goffredo et al., 2010; Pires et al., 2014). Depth is a possible reason for relatively small oocytes in this species. Scleractinians that live in cold temperatures, such as the Antarctic, tend to deposit more lipids in their oocytes during oogenesis as a source of larval nutrition (Waller et al., 2008).

The fourth hypothesis of this study set out to determine if there was a relationship between fecundity and polyp size in terms of height, length and width. In attempts to create a proxy for live coral tissue (as D. dianthus tissue does not extend all the way down the full height of the coral) polyp area (length X height) was used instead of height, length and width. A weak correlation was found between fecundity and polyp area. Indicating the fecundity scales with size slightly but not always. In this study there seemed to be a lot of cases where large individuals happened to not to

30 be fecund or would have low fecundity. No statically significant trend between fecundity and size was found in two other deep-sea scleractinians Lophellia pertusa and Madrepora oculata from the North East Atlantic (Waller & Tyler, 2005).

In this study size of first reproduction is a polyp area of 82 mm in males (coral height: 43.1 mm) and 150 mm in females (coral height: 35.8 mm). It is possible females become reproductive somewhere between a polyp area of 82 and 150 mm however no reproductive females were found smaller then 150 mm in this study. It is also important to note that D. dianthus is relatively slow growing, averaging a growth rate of 2.3 mm yr-1 within the fjord (Häussermann & Förstera 2009). The maximum vertical growth rates for D. dianthus within the fjords is 1.6 mm yr-1. In ideal circumstances with consistent growth (assuming growth is vertical) corals would have to reach a minimum height of 36 mm or an estimated 22.5 years of age before becoming reproductively mature.

The last hypothesis of this study focused on determining differences in reproductive parameters between collection locations. We were hoping to be able to pin point differences in reproduction and location in the study, but due to low sample size this was not possible. Desmophyllum dianthus individuals were statically smaller

(mean height: 34.45 mm; length: 16.67 mm; width: 12.19 mm) at the Punta Huinay collection site compared to the other two sites (Lilihuape: Mean height: 67.28 mm; length: 24.11 mm; width: 16.48 mm. Punta Mamurro mean height: 77.1 mm; length:

23.32 mm; width: 15.34 mm). This could simply be because it is a younger

31 population, thus the population has not had the time to grown and mature like the other two sites.

Environmental stress could also account for the size differences between locations if Punta Huinay is an equally mature site. Punta Huinay is just 400 m from a salmon farming facility where as the other two sites are not in close proximity to fish aquaculture. Salmon farming, especially a facility that has to pump pure oxygen into its pens (Waller, pers. obs.) could be an environmental stress for a sessile benthic organism like D. dianthus. Sedimentation, such as that created from over feeding and concentrated fish waste, can be a significant, even lethal, stress to corals (Rogers,

1983; Rogers 1990; Riegl & Branch 1995). Hypoxia or anoxia may also be taking its toll on the benthic community at the Punta Huinay site (Försterra et al., 2014). In the scope of this study it is unknown as to what is causing the differences between Punta

Huinay and the other two locations. However, I suspect the salmon farm will have an effect on the benthic community at Punta Huinay and further research is needed to assess the situation.

5.1. Summary

This study identified the sex, likely mode of reproduction, the size of first reproduction, and fecundity for Desmophyllum dianthus, a cosmopolitan cold-water scleractinian species. For future research I would like to explore the same reproductive parameters in deeper populations of D. dianthus. It will be intriguing to discover what the oocyte size ranges and maximum oocyte diameters are in the deep

32 populations of D. dianthus. Based on current deep-sea reproductive data one could speculate that oocyte size ranges in deep populations of D. dianthus would not stray drastically from shallower populations but may potentially have a larger maximum oocyte diameter. One could anticipate deeper populations of D. dianthus to be less fecund, similar with what has been seen in Fungiacyathus marenzelleri (Waller et al.,

2002; Flint et al., 2003; Waller & Feehan 2013). In addition to collecting more environmental data from the two fjords focused on in this study, to try and assess to what affect (if any) fish aquaculture is having on benthic coral communities within

Patagonia.

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Waller, R. G., Stone, R. P., Johnstone, J., & Mondragon, J. (2014). Sexual Reproduction and Seasonality of the Alaskan Red Tree Coral, Primnoa pacifica. PloS one, 9(4), e90893.

Wisshak, M., Freiwald, A., Lundälv, T., & Gektidis, M. (2005). The physical niche of the bathyal Lophelia pertusa in a non-bathyal setting: environmental controls and palaeoecological implications. In Cold-Water Corals and Ecosystems (pp. 979-1001). Springer Berlin Heidelberg.

Wu, R. S. S. (1995). The environmental impact of marine fish culture: towards a sustainable future. Marine pollution bulletin, 31(4-12), 159-166. Zakai D., Levy O., & Chadwick-Furman N.E. (2000) Experimental fragmentation reduces sexual reproductive output by the reef-building coral Pocillopora.

Young, C. M. (2003). Reproduction, development and life-history traits. Ecosystems of the World, 381-426.

39 APPENDIX A: SUMMARY OF REPRODUCTIVE DATA BY SITE

Table A.1: Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Lilihuape in the Comau fjord. I.D. Collection Date *d- Depth Height Length Width Sex m-y* (m) (mm) (mm) (mm) 11 10-Aug-12 19.5 86.6 24.9 16.7 N/A 196 10-Aug-12 19.5 114.9 30.3 21.8 F 26 10-Aug-12 19.5 43.5 17.8 13.9 N/A 5 10-Aug-12 19.5 57.7 15.6 12.1 N/A 32 10-Aug-12 19.5 87.2 19.5 14 N/A 6 10-Aug-12 19.5 65.6 17.5 12 M 49 10-Aug-12 19.5 76.6 17.1 13.2 N/A 185 10-Aug-12 19.5 55.1 12.8 11.3 N/A 194 10-Aug-12 19.5 28.8 29.3 19.6 N/A 15 10-Aug-12 19.5 124.7 37.2 21.7 N/A 225 10-Aug-12 19.5 21.5 12.1 9.4 N/A 98 10-Aug-12 19.5 65.9 27.4 17 N/A 38 11-Aug-12 19.5 84.6 23.4 14.5 N/A 190 11-Aug-12 19.5 133 29.5 18 N/A 27 11-Aug-12 19.5 56.7 27.3 16.5 F 64 17-Nov-12 19.5 115.9 24.9 17.6 N/A 197 17-Nov-12 19.5 53.1 12.5 11.8 N/A 19 17-Nov-12 19.5 66.2 23.9 16.6 N/A 85 17-Nov-12 19.5 51.3 21 14.5 N/A 44 17-Nov-12 19.5 65.1 24.1 16 N/A 78 17-Nov-12 19.5 51.2 20.9 14.6 N/A 145 17-Nov-12 19.5 90.5 19.8 18.1 N/A 122 17-Nov-12 19.5 43 10.7 9.2 N/A 93 17-Nov-12 19.5 40 28.5 17.7 N/A 68 17-Nov-12 19.5 15.2 10.3 8.9 N/A 191 17-Nov-12 19.5 92.3 24.5 16.3 F 159 24-Feb-13 19.5 184 38 20 M 59 24-Feb-13 19.5 134.8 28.2 18.8 F 57 24-Feb-13 19.5 47.1 21.7 15.1 F 97 24-Feb-13 19.5 59.3 25.7 18.6 F 188 24-Feb-13 19.5 86.5 21.1 17.3 M

40 Table A.1 (Continued): Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Lilihuape in the Comau fjord. I.D. Collection Date Depth Height Length Width Sex *d-m-y* (m) (mm) (mm) (mm)

34 24-Feb-13 19.5 64.3 26.2 20.2 F 211 24-Feb-13 19.5 44.5 16.6 13.5 N/A 48 24-Feb-13 19.5 28.1 15 11.8 M 171 24-Feb-13 19.5 40.8 31 21.5 F 175 24-Feb-13 19.5 59.1 30 19.6 F 193 24-Feb-13 19.5 59.9 16.9 12.9 N/A 207 24-Feb-13 19.5 20.6 21 16.6 N/A 61 24-Feb-13 19.5 35 30 20.7 F 116 24-Feb-13 19.5 49 19 15.4 M 36 24-Feb-13 19.5 27.8 10 9.5 N/A 104 24-Feb-13 19.5 66.9 16.1 12.5 N/A 218 24-Feb-13 19.5 37.4 17.5 14.7 N/A 89 13-Jun-13 19.5 112.7 24.8 17.5 F 160 13-Jun-13 19.5 104.8 27.7 18.2 M 202 13-Jun-13 19.5 92.6 34.8 18.3 M 108 13-Jun-13 19.5 79.9 28.6 14.7 F 167 13-Jun-13 19.5 33.1 24.5 17.8 M 189 13-Jun-13 19.5 85.1 15.6 12.2 M 129 13-Jun-13 19.5 64.2 22.9 15.5 M 80 13-Jun-13 19.5 54.5 23.6 15.1 M 74 13-Jun-13 19.5 49.3 28.2 19.3 M 67 13-Jun-13 19.5 34.5 17.6 14.5 F 199 13-Jun-13 19.5 44.1 15 8.7 N/A 83 13-Jun-13 19.5 56.1 15 12.1 M 42 22-Sep-13 19.5 127.9 45.1 22.1 N/A 35 22-Sep-13 19.5 123.8 28.2 20.1 N/A 119 22-Sep-13 19.5 76.7 37.2 21.7 N/A 8 22-Sep-13 19.5 54.9 26.1 17.7 N/A 16 22-Sep-13 19.5 63.2 37.7 22.9 N/A 221 22-Sep-13 19.5 46.3 29.2 18.6 N/A 90 22-Sep-13 19.5 67.2 27.2 18.3 N/A

41 Table A.1 (Continued): Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Lilihuape in the Comau fjord. I.D. Collection Date Depth Height Length Width Sex *d-m-y* (m) (mm) (mm) (mm) 161 22-Sep-13 19.5 155.2 29.8 20.7 N/A 150 22-Sep-13 19.5 55.3 20.5 14.9 N/A 124 22-Sep-13 19.5 60.3 31.5 19.1 M 198 22-Sep-13 19.5 45.9 25.5 19.5 N/A 140 22-Sep-13 19.5 33.3 24.2 - N/A 222 22-Sep-13 19.5 16.6 14.1 11.2 N/A 84 22-Sep-13 19.5 34.6 22.8 - N/A 31 22-Sep-13 19.5 29 20.4 - N/A 29 22-Sep-13 19.5 28.7 19.3 - N/A 112 22-Sep-13 19.5 30.5 12 - N/A 30 22-Sep-13 19.5 39 18.8 18.5 N/A 51 22-Sep-13 19.5 121.3 43.5 24.5 N/A 81 22-Sep-13 19.5 38.4 28.3 19.4 N/A 40 22-Sep-13 19.5 103.9 34.9 19.2 N/A 96 22-Sep-13 19.5 37.8 29.4 22.6 N/A 224 22-Sep-13 19.5 72.3 30.8 19.5 N/A 70 22-Sep-13 19.5 30.6 21.6 13.7 N/A

42 Table A.2: Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Punta Huinay in the Comau fjord. I.D. Collection Date Depth Height Length Width Sex *d-m-y* (m) (mm) (mm) (mm) 121 15-Aug-12 25 46.3 17.8 11.9 N/A 18 15-Aug-12 25 25 12 11.2 N/A 21 15-Aug-12 25 40 20.7 14.8 N/A 128 17-Nov-12 25 31.1 17.1 14.2 N/A 134 17-Nov-12 25 38.2 16.2 11.7 N/A 114 17-Nov-12 25 54.3 18.8 14.4 N/A 100 17-Nov-12 25 21.5 14.2 11.9 N/A 148 17-Nov-12 25 27.2 13.5 9.8 N/A 168 17-Nov-12 25 25 18.7 12.8 N/A 130 17-Nov-12 25 48.8 16.8 13.3 N/A 195 17-Nov-12 25 56 18.8 15 N/A 135 17-Nov-12 25 28.3 16.7 14 N/A 53 17-Nov-12 25 22.2 12.2 9.3 N/A 157 17-Nov-12 25 34.2 15.3 10.3 N/A 117 1-Apr-13 25 51.1 20.7 15.15 N/A 9 7-Apr-13 25 33.3 27.4 19.6 N/A 22 7-Apr-13 25 42 21.8 13.3 N/A 215 7-Apr-13 25 33.1 22.7 14.6 M 106 7-Apr-13 25 18.7 18.2 14 M 43 7-Apr-13 25 23.5 17.5 13.6 M 166 7-Apr-13 25 14.3 17.2 13 F 126 7-Apr-13 25 35 19.7 14.4 M 149 7-Apr-13 25 34.9 10.6 9.2 N/A 54 7-Apr-13 25 45.4 16.4 12.5 N/A 217 7-Apr-13 25 10 17.7 11.5 N/A 82 7-Apr-13 25 28.7 15.3 12.4 M 163 7-Apr-13 25 39 21.8 17 N/A 111 7-Apr-13 25 18.3 14.8 13.4 M 174 7-Apr-13 25 19.2 11.9 9.1 N/A 210 7-Apr-13 25 13.1 7 5.9 N/A

43 Table A.2 (Continued): Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Punta Huinay in the Comau fjord. I.D. Collection Date Depth Height Length Width Sex *d-m-y* (m) (mm) (mm) (mm) 71 13-Jun-13 25 41.8 17.8 13.3 F 65 13-Jun-13 25 47.2 20.9 15.6 N/A 179 13-Jun-13 25 33 15.8 11.3 M 143 13-Jun-13 25 24.8 18.1 13.7 M 182 13-Jun-13 25 17.2 13.4 10.2 M 109 13-Jun-13 25 26.4 11.8 9.1 N/A 139 13-Jun-13 25 19.1 9 7.6 N/A 113 13-Jun-13 25 56.7 19.8 2.3 N/A 10 13-Jun-13 25 25.1 14.6 11.2 F 192 13-Jun-13 25 28 8.1 7.6 N/A 186 21-Sep-13 25 57.5 23.9 16.4 N/A 165 21-Sep-13 25 34 16.8 12.7 N/A 69 21-Sep-13 25 36.2 13.8 10.9 N/A 103 21-Sep-13 25 37 10.4 9.9 N/A 23 21-Sep-13 25 47.8 22.1 15.6 N/A 75 21-Sep-13 25 43 24.3 15.9 N/A 56 21-Sep-13 25 43.1 10.5 7.8 M 66 21-Sep-13 25 51 14.9 10.8 N/A 181 21-Sep-13 25 31.2 15.6 11.5 N/A 212 21-Sep-13 25 50.9 12.9 10.4 N/A 107 21-Sep-13 25 56.7 18.2 12.1 N/A 209 21-Sep-13 25 29.5 16.1 12.2 N/A 155 21-Sep-13 25 36.2 18.8 12.9 N/A 95 21-Sep-13 25 34.1 19.4 13.5 N/A 92 21-Sep-13 25 30 20.5 13 N/A

44 Table A.3: Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Punta Mammuro in the Renihue fjord. I.D. Collection Date Depth Height Length Width Sex *d-m-y* (m) (mm) (mm) (mm) 1 13-Aug-12 27 106 23.2 15.1 N/A 2 13-Aug-12 27 102 25.7 16.7 M 3 13-Aug-12 27 66 22.4 19.4 N/A 4 13-Aug-12 27 170 30.9 17.5 M 33 13-Aug-12 27 73 20.5 13.9 N/A 206 13-Aug-12 27 92 33 21.3 N/A 158 13-Aug-12 27 148.9 38.5 23.3 M 170 13-Aug-12 27 182.6 19.5 14.1 M 13 13-Aug-12 27 71.5 12.5 11.3 N/A 37 13-Aug-12 27 99 46.7 19.9 N/A 208 13-Aug-12 27 125.5 25.5 15.8 M 147 13-Aug-12 27 58.3 17.2 14.4 N/A 88 13-Aug-12 27 37.4 11.8 9.8 N/A 14 13-Aug-12 27 60.9 22.8 16.1 N/A 136 13-Aug-12 27 64.2 15.6 13.1 N/A 76 21-Sep-12 27 104.1 19.3 14 N/A 138 21-Sep-12 27 71.6 14.9 12.1 N/A 120 21-Sep-12 27 90.3 15 12.2 N/A 205 21-Sep-12 27 86.1 22.3 15.3 N/A 200 21-Sep-12 27 90.1 20 12.6 N/A 52 21-Sep-12 27 106.8 19.2 13.9 N/A 154 21-Sep-12 27 56.5 21.2 14 N/A 183 21-Sep-12 27 27.5 25.1 16.8 N/A 20 19-Mar-13 27 63.3 15.8 13 N/A 156 19-Mar-13 27 129 26.5 13.9 M 73 19-Mar-13 27 86.25 32.1 19.4 F 28 19-Mar-13 27 101.3 22.5 16.1 M 101 19-Mar-13 27 69.8 25.2 15.7 M 177 19-Mar-13 27 66.2 36.9 19.8 M 176 19-Mar-13 27 139.5 32.9 17.9 M 17 19-Mar-13 27 19.7 16.5 11.5 N/A 46 19-Mar-13 27 35.8 28.4 19 F 24 19-Mar-13 27 24.5 19.6 11.8 M 25 19-Mar-13 27 41.9 16 11.5 M 105 19-Mar-13 27 29 11 10.3 N/A 173 19-Mar-13 27 35.8 13 11.5 F

45 Table A.3 (Continued): Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Punta Mammuro in the Renihue fjord. I.D. Collection Date Depth Height Length Width Sex *d-m-y* (m) (mm) (mm) (mm) 7 19-Mar-13 27 30.7 11.5 9.5 N/A 203 19-Mar-13 27 30 12.2 9.7 N/A 184 20-Sep-13 27 106.5 36.7 20.3 N/A 45 20-Sep-13 27 86.5 40.3 21.6 N/A 72 20-Sep-13 27 65.9 28.9 20.1 N/A 91 20-Sep-13 27 63.9 33.4 18.5 N/A 12 20-Sep-13 27 45.1 28.1 18.7 N/A 79 20-Sep-13 27 31.8 15.9 12.6 N/A

Table A.4: Summary of size parameters and sex of Desmophyllum dianthus individuals collected at Cabudahue in the Renihue fjord. I.D. Collection Date Depth Height Length Width Sex *d-m-y* (m) (mm) (mm) (mm) 164 13-Aug-12 18.3 44.8 21.9 16.4 N/A 204 13-Aug-12 18.3 64.4 37.3 24.3 N/A 62 13-Aug-12 18.3 40.9 27.7 19.5 F 219 13-Aug-12 18.3 71.2 32.3 21.1 N/A 99 13-Aug-12 18.3 56.9 21.3 15.1 N/A 125 13-Aug-12 18.3 46.3 25.5 16.2 N/A 58 13-Aug-12 18.3 43.2 21.3 17.7 N/A 187 13-Aug-12 18.3 34.2 20.3 12.8 N/A 110 13-Aug-12 18.3 57.3 19.7 15.3 N/A 223 13-Aug-12 18.3 58.4 20.2 15.8 N/A 131 13-Aug-12 18.3 74.4 36.6 20 N/A 87 13-Aug-12 18.3 64.6 38.9 22.2 N/A 47 13-Aug-12 18.3 26.8 26.7 17.8 N/A 169 13-Aug-12 18.3 37.4 32.1 22.7 F 146 13-Aug-12 18.3 34.1 23 18 M 220 13-Aug-12 18.3 47 24.9 17.3 N/A 63 13-Aug-12 18.3 40 21.4 16.9 N/A

46 APPENDIX B: SUMMARY OF FECUNDITY DATA

Table B.1: Summary of fecundity (opp), average oocyte diameter, polyp area and number of reproductive mesenteries in females from all locations. ID Fecundity STD SE Polyp Location Month Average # of (opp) Area Oocyte Repro. Diameter Mesent (um) eries 191 10816 24.09 13.91 399.35 Lilihuape Nov '12 57.293 78 34 172328 179.57 103.67 529.24 Lilihuape Feb '13 124.894 78 171 161486 60.47 34.91 666.5 Lilihuape Feb '13 131.947 78 166 8662 12.00 6.93 223.6 Punta Huinay Apr '13 150.469 71 67 43628 61.08 35.26 255.2 Lilihuape Jun '13 183.63 78 124 33800 28.57 16.50 601.65 Lilihuape Sep '13 50.922 78 108 135330 166.52 96.14 420.42 Lilihuape Jun '13 192.905 78 59 122265 130.61 75.41 530.16 Lilihuape Feb '13 124.325 78 73 147240 364.59 163.05 622.74 Punta Mamurro Mar '13 176.382 45 97 162552 349.03 201.51 478.02 Lilihuape Feb '13 150.097 78 10 7975.666 13.80 3.83 163.52 Punta Huinay Jun '13 201.609 71 46 10305 12.29 7.09 539.6 Punta Mamurro Mar '13 148.973 45 175 59332 131.11 75.70 588 Lilihuape Feb '13 139.504 78 71 8212.333 33.84 19.54 236.74 Punta Huinay Jun '13 178.638 71 89 55302 183.66 106.04 434 Lilihuape Jun '13 190.359 78 57 75270 354.06 204.41 327.67 Lilihuape Feb '13 142.341 78 173 88305 256.29 147.97 149.5 Punta Mamurro Mar '13 178.638 45 169 6684 37.55 21.68 728.67 Cabudahue Aug '12 214.546 36 27 29068 153.21 88.45 450.45 Lilihuape Aug '12 309.505 78 196 60502 38.08 21.99 660.54 Lilihuape Aug '12 292.28 78 61 163254 120.68 69.67 621 Lilihuape Feb '13 124.675 78 62 2448 8.89 5.13 540.15 Cabudahue Aug '12 267.596 36

APPENDIX C: SUMMARY OF COLD-WATER SCLERACTINIAN REPRODUCTIVE DATA

Table C.1: Summary of sexuality, mode of reproduction and maximum oocytes sizes in all deep-sea Scleractinians published to date. Species Area Ocean/ Depth Max Reference Sea (m) Oocyte (µm) Caryophyllia Porcupine N. 435- 340 Waller et al., cornuformis Seabight Atlantic 2000 2005 (Pourtales 1868) Caryophyllia Porcupine N. 1100- 630 Waller et al., ambrosia (Alcock Seabight Atlantic 3000 2005 1898) Caryophyllia Porcupine N. 960- 450 Waller et al., seguenzae Seabight Atlantic 1900 2005 (Duncan 1873) Caryophyllia Elba Mediterra 40- 141 Goffredo et al., inornata (Duncan Island nean 526 2012 1878) Caryophyllia Plymouth N. 150 Tranter et al., smithi (Lamarck Breakwat Atlantic 1982 1801) er Lophelia pertusa Porcupine N. 900 140 Waller & Tyler, Seabight Atlantic 2005 Lophelia pertusa Campos S.W. 600 337 Pires et al., Basin - Atlantic 2014 Brazil Lophelia pertusa Trondhei Norweigi 40-500 180 Brooke & m Fjord an Sea Järnegren, 2013 Solenosmilia Chatham Pacific 800- 165 Burgess & variabilis Rise 1000 Babcock, 2005 (Duncan, 1873) Solenosmilia Campos S.W. 600 242 Pires et al., variabilis Basin - Atlantic 2014 Brazil Goniocorella Chatham Pacific 800- 135 Burgess & dumosa (Alcock, Rise 1001 Babcock, 2005 1902) Astroides Palinuro Mediterra 40-368 1590 Goffredo et al., calyculris (Pallas, Italy nean 2010 1766) Enallopsammia Chatham Pacific 800- 400 Burgess & rostrata (De Rise 1000 Babcock, 2005 Pourtales, 1878) Enallopsammia Campos S.W. 600 1095 Pires et al., rostrata Basin - Atlantic 2014 Brazil Leptopsammia Eastern Mediterra 15- 680 Goffredo et al., pruvoti (Lacaze- Ligurian nean 16.5 2006 Duthiers) Sea Tubastraea Equatorial E. Pacific 2-5m 1000 Glynn et al., coccinea (Lesson, Eastern 2008 1829) Pacific

Table C.1 (Continued): Summary of sexuality, mode of reproduction and maximum oocytes sizes in all deep-sea Scleractinians published to date. Species Area Ocean/ Depth Max Reference Sea (m) Oocyte (µm) Flabellum N.W. N. 1430 1200 Mercier et angulare Atlantic Atlantic al., 2011 (Moseley, 1876) Flabellum Porcupine N. 2412- 1015 Waller & angulare Seabight Atlantic 2467 Tyler, 2011 Flabellum Western Souther 500 4800 Waller et al., thouarsii Antarctic n 2008 Peninsula Flabellum Western Souther 500 5120 Waller et al., curvatum Antarctic n 2008 Peninsula Flabellum Western Souther 500 5167 Waller et al., impensum Antarctic n 2008 Peninsula Fungiacyathus Rockall N. 2000 750 Waller et al., marenzelleri Trough Atlantic 2002 (Vaughan, 1906) Fungiacyathus California N. 4100 750 Flint et al., marenzelleri Pacific 2003 Fungiacyathus Western Souther 600 1400 Waller & marenzelleri Antarctic n Feehan, Peninsula 2012 Madrepora Porcupine N. 900 350 Waller & oculata Seabight Atlantic Tyler, 2005 (Linnaeus 1758) Madrepora Chatham Pacific 800- - Burgess & oculata Rise 1000 Babcock, 2005 Madrepora Campos S.W. 600 650 Pires et al., oculata Basin - Atlantic 2014 Brazil Oculina Florida N. 3-100 100 Brooke 2003 varicosa Atlantic (Lesueur 1821)

49 BIOGRAPHY OF THE AUTHOR

Keri Feehan was born in Leominster, Massachusetts on May 13th 1991. She was raised in Lunenburg, Massachusetts and graduated from Lunenburg High School in 2009. She attended the University of Maine Fall 2009. In 2012, Keri received the Dearborn Scholarship for a summer internship at the Ira C. Darling Marine Center under the advisement of Dr. Rhian Waller. The research she participated in during 2012 was then published in 2013 – her senior year (Waller, R.G. and Feehan, K.F. 2013. Reproductive Ecology of a Polar Deep-Sea Scleractinian Fungiacyathus marenzelleri (Vaughan, 1906). Deep-Sea Research Part II). In addition, Keri was indicted into the Phi Beta Kappa Honors Society in May 2013. Keri graduated from the University of Maine with a Bachelor of Science in Marine Science in 2013. After graduation she continued to work on her senior capstone project at the Ira C. Darling Marine Center in the Waller Laboratory. In January 2014, she began her Master’s Degree at the University of Maine under the advisement of Dr. Rhian Waller. In 2015, the research she started as her undergraduate senior capstone project and continued thereafter, was published (Feehan, K.A. and Waller, R.G. 2015. Notes on the Reproduction of Eight Species of Eastern Pacific Cold-Water Octocorals. Journal of the Marine Biological Association). After receiving her degree Keri will start a teaching position at Lincoln Academy in Newcastle, Maine. Keri is a candidate for the Master of Science degree in Marine Biology from the University of Maine in May 2016.