FINAL REPORT TO THE MASTS VISITING FELLOWSHIP SCHEME

Role of environmental cues in driving variability of reproductive periodicity in North Atlantic deep-water corals

Sandra Brooke, Florida State University

J. Murray Roberts, Heriot-Watt University

December, 2014

Abstract This project focuses on the reproductive biology of the structure-forming cold-water coral Lophelia pertusa. This species is widely distributed but is particularly abundant in the North Atlantic, especially off the UK and Norway, and off the southeastern US and Gulf of Mexico. Over the past decade, various research efforts have facilitated the collection of samples of L. pertusa from these different locations. Research into the reproduction of this species has shown regional differences in the timing of gametogenic cycles, with the western Atlantic populations spawning several months earlier than those in the eastern Atlantic. The causes of these differences still need to be resolved but preliminary data show they are probably related to the timing of food supply. Samples of L. pertusa were collected from off Scotland between 2009 and 2012, and were archived by Prof. Roberts at Heriot-Watt University. Through the MASTS visiting fellows program, Dr Brooke (Florida State University, USA) worked with Prof. Roberts to process these archived samples, which show similar characteristics to other populations in the NE Atlantic. These data will be incorporated into the first basin-scale study of deep-sea coral reproduction. Environmental and biotic data will be compared across regions to identify the factors that are driving the observed regional differences in gametogenic cycles. Introduction Over the past decade or more, deep-water corals and the threats they face have come to the forefront of scientific and political interest. Their distribution is extensive, and their ecological value is significant. Many species of deep-sea coral provide structure that supports an abundant and diverse community of invertebrates and fish. Unfortunately large areas of coral habitat have been damaged by mobile fishing gear, including those constructed by the hard coral Lophelia pertusa. This coral is found in most of the world’s oceans and is particularly abundant in the North Atlantic. Although science has made great progress in deep-water coral research in recent years, there are still significant gaps in our understanding of coral biology and ecology with studies lacking comprehensive or targeted data collection. This MASTS fellowship project focused on the reproductive biology of the widely distributed, reef-building deep-water coral Lophelia pertusa. Initial observations on reproduction in this species were published in 2005 (Waller and Tyler), but the geographic and temporal spread of the samples were limited. Since 2005, many additional samples of L. pertusa and other deep-water corals have been collected from both eastern and western Atlantic, and a more comprehensive study of the reproduction of this species was published (Brooke and Jarnegren 2013) using samples from the Trondheim Fjord (Norway). The goal of this fellowship was to process and analyse samples of L. pertusa from the northeastern Atlantic, which were housed with Dr. Roberts at Heriot-Watt University, and incorporate those data into an existing database from the western Atlantic (owned by Dr Brooke) to create the first basin-scale study of reproduction in a deep-water coral. The oceanographic and environmental conditions differ between coral collection sites in the northeastern and western Atlantic, especially in the seasonality of primary productivity and delivery of organic material to the seafloor. This and other variables such as temperature, current regime and tidal or diurnal signals may influence timing of reproduction in our target corals. By comparing the timing of reproductive cycles of deep-water corals that live under different conditions, we can investigate the effects of exogenous factors on the reproductive periodicity of these ecologically significant species. In addition to their sample inventory, the partners on this project also have long-term, high-resolution environmental data from their coral study sites, acquired from instruments deployed

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on benthic landers and moorings. The combination of spatially and temporally comprehensive coral collections across the North Atlantic basin, and the long term environmental information from the study sites creates a truly unique and valuable data series. Results from this work will have implications for other deep-sea fauna, thus increasing the value of this research. Methods Sample collections Samples of L. pertusa were collected during five separate research cruises in 2009 (RSS Discovery in June-July), 2010 (RV Calanus in February), 2011 (RSS Discovery in June-July and RV Poseidon in September) and 2012 (RRS James Cook in May-June). Collections were made either using a video guided grab, ROV or research submersible, depending on the cruise. The locations of these cruises are shown in figure 1 and a summary of samples processed and their location data is provided in table 1. Figure 1: Map of sites sampled during five research cruises from 2009-2012 (Image: Google Earth)

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Table 1: Summary of samples collected showing sample code name and location information

Sample # Date Site name Latitude (GPS)

Longitude (GPS)

Depth (m)

M1 May 1, 2011 Murchison No data No data 155 M2-M4 April 28, 2011 Ninian North No data No data 100

M5 July 7, 2011 Mingulay 56 49.569 -7 23.475 169 M6 July 7, 2011 Mingulay 56 49.325 -7 23.855 142 M7 July 7, 2011 Mingulay 56 49. 503 -7 23.601 159 M8 July 7, 2011 Mingulay 56 49.569 -7 23.475 169 M9 July 7, 2011 Mingulay 56 49.321 -7 24.040 135 M10 July 7, 2011 Mingulay 56 49.262 -7 24.173 121 M11 July 7, 2011 Mingulay 56 49. 365 -7 23.809 137 M12 July 7, 2011 Mingulay 56 49.419 -7 23.928 134 M13 July 7, 2011 Mingulay 56 49.446 -7 23.044 154 M14 July 7, 2011 Mingulay 56 49.480 -7 23.550 143 M15 July 7, 2011 Mingulay 56 49.309 -7 23.904 133 M16 June 8, 2011 Mingulay 56 49.284 -7 23.059 142 M17 June 8, 2011 Mingulay 56 49.221 -7 23.006 164 M18 June 8, 2011 Mingulay 56 49.221 -7 23.006 164 M19 June 8, 2011 Mingulay 56 49.089 -7 23.187 146 M20 June 8, 2011 Mingulay 56 49.221 -7 23.006 164

M20A June 8, 2011 Mingulay 56 49.284 -7 23.059 146 M21 June 8, 2011 Mingulay 56 49.158 -7 23.085 124 M22 June 8, 2011 Mingulay 56 49.269 -7 23.492 154 M23 June 8, 2011 Mingulay 56 49.089 -7 23.187 146 M24 June 8, 2011 Mingulay 56 49.145 -7 23.032 167 M25 June 8, 2011 Mingulay 56 49.338 -7 23.587 164 M26 June 8, 2011 Mingulay 56 49.158 -7 23.085 124 M27 June 8, 2011 Mingulay 56 49.338 -7 23.587 164 M28 June 8, 2011 Mingulay 56 49.021 -7 23.575 150 M29 June 8, 2011 Mingulay 56 49.221 -7 23.006 114 M30 June 8, 2011 Mingulay 56 49.172 -7 23.557 131 M31 Feb. 23, 2010 Mingulay 56 49.397 -7 23.859 128 M32 Feb. 23, 2010 Mingulay 56 49.403 -7 23.707 130 M33 Feb. 23, 2010 Mingulay 56 49.323 -7 23.925 135

M34-M42 Sept. 20, 2011 Nordleska 63 36.40 -9 23.36 160-187 M43 May 22, 2012 Mingulay 1 56 49.375 -7 23.697 134 M44 May 22, 2012 Mingulay 1 56 49.375 -7 23.697 134 M45 May 22, 2012 Mingulay 1 56 49.360 -7 23.717 134 M46 June 6, 2012 Logachev-1 55 33.669 -15 39.327 No data M47 June 7, 2012 Pisces 9 57 36.599 -14 29.545 259 M48 June 4, 2012 Logachev-3 55 33.060 -15 47.220 770 M49 May 30, 2012 Logachev-2 55 29.654 -15 49.327 844 M50 June 7, 2012 Pisces 9 57 36.588 -14 29.519 258

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M51 May 22, 2012 Mingulay 1 56 49.356 -7 23.712 134 M52 June 4, 2012 Logachev-3 55 33.060 -15 47.220 770 M53 May 22, 2012 Mingulay 1 56 49.375 -7 23.697 134 M54 June 6, 2012 Logachev-1 55 33.669 15 39.327 No data M55 June 7, 2012 Pisces 9 57 36.588 14 29.519 258 M56 June 8, 2012 Rockall Bank 57 57.716 13 59.589 221 M57 June 7, 2012 Pisces 9 57 36.588 14 29.518 258 M58 June 7, 2012 Pisces 9 57 36.588 14 29.518 258 M59 June 4, 2012 Logachev-3 55 33.609 15 47.055 696 M60 June 7, 2012 Pisces 9 57 36.540 14 29.467 258 M61 June 7, 2012 Pisces 9 57 36.528 14 29.413 258 M62 July 1, 2009 Banana Reef 56 48.234 7 26.736 138 M63 July 1, 2009 Banana Reef 56 48.330 7 26.520 127 M64 July 1, 2009 Banana Reef 56 48.384 7 23.430 127 M65 July 1, 2009 Banana Reef 56 48.090 7 27.198 165 M66 July 1, 2009 Mingulay 56 49.212 7 23.568 108 M67 July 1, 2009 Mingulay 56 49.368 7 23.670 127 M68 July 3, 2009 Mingulay 56 49.440 7 23.826 146 M69 July 1, 2009 Mingulay 56 49.314 7 23.838 137 M70 July 1, 2009 Mingulay 56 49.374 7 23.700 127 M71 July 3, 2009 Mingulay 56 49.386 7 23.682 131 M72 June 28, 2009 Mingulay 56 49.382 7 23.69985 127 M73 July 1, 2009 Mingulay 56 49.372 7 23.702 127 M74 July 2, 2009 Mingulay 56 49.395 7 23.73248 134

Sample processing Samples were fixed using 5% formalin on board ship then transferred to 70% ethanol for long term storage. The samples were rinsed briefly in distilled water then decalcified using 10% hydrochloric acid solution to dissolve the coral skeleton and expose the tissue. The samples were then dehydrated through a series of ethanol concentrations (2 days per batch) and placed into a clearing agent (toluene or Histoclear©) for 24 hours. The samples were then soaked in melted paraffin wax for 24-48 hours and transferred to small trays to set. These blocks of paraffin with embedded tissue were then cut in 8 µm sections and mounted onto microscope slides. When dry, the slides were stained using Haematoxylin and Eosin stains (to highlight DNA and lipids respectively) and sealed with a cover slip. Serial images were taken of all the microscope slides using a camera attached to a compound microscope. These images were calibrated using an ocular micrometer, and image analysis software (Digimizer©) was used to measure the area of ~100 different oocytes from each female sample. Mean oocyte ferret diameter (calculated from the area measurements) measurements were generated for each sample. The male reproductive status was staged according to maturity of gamete development. Results A total of 74 samples were processed; of these 24 were female, 15 were male and the remainder either had no gametogenic material or gender could not be determined as the germ cells were immature. Early germ cells were observed in samples from late April, but female gametes were

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found in those from June (mean = 29.83, SD = 6.15; n=4), July (mean = 35.52, SD = 5.12; n= 7) and September (mean = 53.56, SD = 11.30; n= 1). The male gametogenic cycle was documented by stages (after Waller and Tyler 2005) as follows: Stage I (early spermatogenesis); Stage II (maturation phase); Stage III (mature) and Stage IV (post-spawn). June and July samples were early stage II and in September the spermatocysts were in the maturation phase (stage II-III) with darkly stained spermatocytes. Examples of different female and male reproductive stages are shown in figure 2A-F, and the distribution of mean oocyte diameters are shown in figure 3. Figure 2: Images of sections of Lophelia pertusa showing different time periods and stages of gametogenesis: A) early gametogenesis showing dark germ cells collected in the mesoglea; B) early vitellogenesis, oocytes are approximately 28 µm in diameter; C) samples from July showing more mature oocytes (35-40 µm diameter); D) Oocytes in September are larger (50 – 60 µm diameter); E) images of male spermacysts from samples collected in June, showing dense spermatocytes; F) more mature spermatocysts from samples collected in September.

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Figure 3: Changes in mean oocyte diameter (µm) of Lophelia pertusa samples from the project study sites over time

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Discussion There was an insufficient time series of samples to completely elucidate the gametogenic cycle of L. pertusa in the study region; however, oocyte sizes from these samples were compatible with the more complete gametogenic cycles described from Trondheim Fjord populations. The corals were observed spawning in the Trondheim populations in late January to early March, and the samples from this project were spent (spawned) in late February with early germ cells appearing in the spring (April). This differed from the Trondheim populations as the latter showed overlapping cycles with early gametes co-occurring with mature eggs and sperm, but was similar to observations by Waller and Tyler (2005), who studied North Sea populations of Lophelia and did not observe overlapping cycles. This is an interesting difference and may be related to food supply differences between fjord and open areas of the northeastern Atlantic. The oocyte diameters from the project samples from June (mean = 29.83, SD = 6.16) and September (mean = 53.56, SD = 11.30) overlapped in size with those from the same time periods from the Trondheim Fjord (mean = 28.01, SD = 7.62 for June; mean = 57.83, SD = 9.38 for September) . The L. pertusa populations from the western Atlantic are on a different gametogenic schedule from the eastern Atlantic populations (Brooke et al. 2007, Demopoulos et al. in press, Brooke unpub data). Spawning was observed in late September for specimens collected from the Gulf of Mexico (Brooke pers. obs.) and is inferred (from histological sections, Brooke unpubl data) to occur in late October in the southeastern US populations. This represents a temporal offset of approximately 4 months, with the western Atlantic populations spawning before the eastern

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Atlantic. Comparable data from conspecific populations under different regional environmental regimes provides an unusual opportunity to investigate the factors that may be driving reproductive processes. An examination of long term-temperature, oxygen and current speeds for each region does not show any apparent correlations with aspects of the coral reproductive cycles. The most promising factor so far is food supply; the timing of primary productivity in the photic zone can be readily derived from satellite imagery and a great deal of published literature. Information on timing of organic material delivery to the seafloor is sparse but in the northeastern Atlantic the spring peak occurs in June-July with a smaller autumn peak. In the western Atlantic the spring peaks occur approximately 3 months earlier (March-April), which almost mimics the difference in reproductive cycles. The secondary production of zooplankton, (and other variables) may complicate this simplistic view, and more research needs to be done to determine the mechanisms through which biotic and abiotic factors control reproductive processes in these deep sea species. The data generated through the MASTS fellowship with be incorporated into the database we are developing on reproduction of L. pertusa and other deep-sea corals. The final outcome of this research will be a comprehensive manuscript on basin-wide reproductive ecology of this important structure-forming species, incorporating the work done through the MASTS fellowship. Literature cited Brooke S., J. Jarnegren (2013) Reproductive periodicity of the deep-water scleractinian coral, Lophelia pertusa from the Trondheim Fjord, Norway. Mar. Biol.160:139-153 Brooke S., C.M. Young, M. Holmes (2007) Chapter 6: Biological Characterization and Studies, In: Characterization of Northern Gulf of Mexico deepwater hard bottom communities with emphasis on Lophelia coral. Final report to U.S.D.I., Minerals Management Service, Gulf of Mexico Region. OCS Study 2007-044: p119-147 Demopoulos A.W.J., S.W. Ross, S. Brooke, et al. (In press) Deepwater Program: Lophelia II Continuing Ecological Research on Deep-Sea Corals and Deep Reef Habitat in the Gulf of Mexico. US Geological Survey Report. Waller R.G., P.A. Tyler (2005) The reproductive biology of two deep-water reef-building scleractinians from the NE Atlantic Ocean. Coral Reefs 24: 514-522

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Reproductive cycles of Lophelia pertusa in the North Atlantic

Sandra Brooke, Florida State University

J. Murray Roberts, Heriot-Watt University

Mingulay reef

• Describe timing of reproductive cycles of Lophelia pertusa from Mingulay reef

• Compare with reproduction of conspecifics from

elsewhere in the North Atlantic Ocean

• Identify potential drivers of reproduction using

environmental and biological variables

Objectives

Gametogenesis in Lophelia pertusa

February

September July June

April

~30 microns ~35 microns ~55 microns

Calendar Week

4 8 12 16 20 24 28 32 36 40 44 48 52

Ooc

yte

diam

eter

( m

)

0

10

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30

40

50

60

70Changes in mean oocyte diameter over time

Spawned: Early germ cells

Trondheim Fjord, Norway

Tautra

Stokkbergneset

Roberg

Changes in mean oocyte diameter over time: NE Atlantic

Brooke and Jarnegren 2013 Month

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m)

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J M F A J M A J S O N D

Spawning

Gulf of Mexico, and southeastern US

Changes in mean oocyte diameter over time Gulf of Mexico and Southeastern US

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J F M A M J J A S O N D

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GOMSEUS

Spawning ? Spawning

Changes in mean oocyte diameter over time: all regions combined

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J F M A M J J A S O N D

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m)

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Norway GOM SEUS

Spawning (estimated)

Spawning (observed)

Spawning (observed)

Mingulay

Drivers of shallow coral reproduction

Seasonal changes in: •Seawater temperature •Current and/or wind patterns •Solar insolation/daylength •Lunar and/or tidal cycles •Energy supply (egg maturation or larval food) •Internal processes (e.g. hormone levels)

Timing organic material delivery to the seafloor

NE Atlantic: June-July (with a smaller autumn peak)

GoM: March-April (with other peaks during the year)

SEUS: Spring peak (confounded by upwelling productivity)

NE Atlantic vs. GoM

Spawning: 4-5 months difference

Food peaks: 3-4 months difference

Food supply can influence reproductive cycles in the deep sea

Summary

NE Atlantic, GoM and SEUS populations are offset by several months

Food supply correlates more closely to reproductive cycles in each location, but does not fit exactly

Environmental variables (available data) cannot explain the observed differences in reproductive cycles

Timing of reproduction is important in the context of oceanography, as it influences connectivity between regions

Applications to management

• Information on life history processes is important information for any managed species

• Timing of spawning periods and duration of larval life can be combined with oceanographic models to predict regional connectivity

• Reproductive processes may change in response to increased stress. Baseline data is essential for assessing impacts of natural and anthropogenic stressors.

• Management actions can avoid potentially damaging activities during peak reproductive periods