Exploring the Life Histories of Cephalopods Using Stable

EXPLORING THE LIFE HISTORIES OF CEPHALOPODS USING STABLE

ISOTOPE ANALYSIS OF AN ARCHIVAL TISSUE

KIRT LEE ONTHANK

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

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY School of Biological Sciences

MAY 2013 To The Faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of KIRT LEE ONTHANK find it satisfactory and recommend that it be accepted.

______Raymond W. Lee, Ph.D., Chair

______Asaph B. Cousins, Ph.D.

______David F. Moffett, Ph.D.

ii ACKNOWLEDGEMENTS

This dissertation, and in fact my career as a scientist, is the result of innumerable contributions from many people who I can only offer here thanks weak and meager compared to what you are owed.

First, I thank advisor, Ray Lee. Your patience with my unworkable research ideas and slow, sloppy writing; your guidance in navigating grad school and peer-review have made this work possible. My greatest regret from my time at WSU is not drawing more from the depth of your wisdom, knowledge, and insight. I sincerely hope I can rectify that in the years to come.

Thank you to my committee: David Moffett and Asaph Cousins. Thank you for being patient with my poor communication, and my misuse of my committee. Thank you for pushing me to produce work that I can be proud of.

I need to thank my former professors at Walla Walla University: David Cowles, Jim

Nestler, Joe Glusha, Scott Ligman, and Gene Stone. I thank you for believing in me before I did, for treating me as a colleague long before I had earned it, and for not only tolerating this loud, awkward, overly-confident young man from the outside, but also entrusting me with some measure of responsibility.

I must thank my parents who raised me in a house in which wonder of the natural world, especially living things, was part of the fabric of daily life. I thank you for allowing me to lose snakes in my bedroom, for showing me red giants and fuzzy nebulae in the night sky, for filling our garage with geckos, tarantulas, stick insects, tortoises, scarab beetles, and an iguana, for sitting silently with me in a central Idaho clear-cut just to see what would walk out of the forest as night fell, for buying me copper sulfate that you would sweep from behind the fridge years

iii later, for introducing me to Carl Sagan, David Attenborough, and Marty Stouffer, for helping me to succeed, and allowing me to fail. Without your love and support it is unthinkable that I could be the person I am today.

Finally, to my wife, Stephanie, I offer here all the thanks my feeble words can muster, and hope my actions in person can more fully expresses my gratitude. You helped me rediscover my childhood dream of becoming a scientist, changing the course of my life forever. When pursuit of that dream led to long, repeated absences as I pursued octopuses at Rosario, dove in ALVIN while at sea for weeks, or just gone for another week in Pullman, your support never flagged.

When I reached my thirtieth birthday and was still a student, your patience did not fail. I would not be me if I had never met you, and for that I owe you everything I will be.

iv EXPLORING THE LIFE HISTORIES OF CEPHALOPODS USING STABLE

ISOTOPE ANALYSIS OF AN ARCHIVAL TISSUE

Abstract

ABSTRACT by Kirt Lee Onthank, Ph.D. Washington State University May 2013

Chair: Raymond W. Lee

Relatively little is known about the life histories of cephalopods compared to many other groups of major marine predators such as fish, marine mammals, and sea birds. Increased importance of cephalopods to global fisheries in the past forty years and a recognition of the important ecological roles of cephalopods has driven increased research interest into the lives of these enigmatic animals. Still, there is a paucity of information about the life histories of all cephalopods except a few well-studied species.

Stable isotope analysis has become a powerful tool to infer nutritional sources and movement patterns in aquatic organisms. The use of this tool can be extended temporally when used in tissues that have no elemental turnover after formation, or archival tissues, which provide a frozen record of stable isotope composition at formation. In this dissertation I explore the utility of using stable isotope analysis of an archival tissue, eye lens, in cephalopods to help provide sorely needed information about the life histories of this ubiquitous class of marine mollusks.

Here the use of stable isotope analysis of eye lenses is used to investigate possible natal

v origins of Dosidicus gigas from the northeastern Pacific ocean. This species of large squid has recently undergone a dramatic northward range expansion, pushing the northern extent of occurrence from 32ºN before 1997 to 55ºN by 2008. Data presented suggests squid collected in this study from the coastal US were hatched north of 37ºN. Additionally, eye lens isotope data is used to confirm dietary data from midden analysis of two north Pacific octopuses, Enteroctopus dofleini and Octopus rubescens. This data helps identify these octopus species as potentially providing the predation pressure to maintain a mutualism between a sponge and a scallop.

Finally, stable isotope analysis of the eye lens of a hydrothermal vent-associated octopus,

Graneledone cf. boreopacifica, demonstrates that this octopus likely did not consume any hydrothermal vent fauna over the course of its lifetime, despite being collected near productive vent habitats. Together these chapters show the utility of this method to help elucidate life histories of cephalopods.

vi TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... iii

ABSTRACT...... v

TABLE OF CONTENTS...... vii

LIST OF TABLES...... ix

LIST OF FIGURES...... x

CHAPTER ONE GENERAL INTRODUCTION...... 1

REFERENCES...... 9

CHAPTER TWO CARBON AND NITROGEN STABLE ISOTOPE ANALYSIS OF EYE LENSES SUGGEST NATAL ORIGINS AND MIGRATORY HISTORIES OF NORTHWARD RANGE EXPANDING HUMBOLDT SQUID (DOSIDICUS GIGAS)...... 18

ABSTRACT...... 19

INTRODUCTION...... 20

MATERIALS AND METHODS...... 23

RESULTS...... 26

DISCUSSION...... 28 Diet...... 28 Migration...... 31 REFERENCES...... 36

CHAPTER THREE A SPONGE-SCALLOP MUTUALISM MAY BE MAINTAINED BY OCTOPUS PREDATION...... 52

ABSTRACT ...... 53

INTRODUCTION...... 54

vii MATERIALS AND METHODS...... 58 Determination of Diet...... 58 Preference Trials...... 61 Statistics...... 63 RESULTS...... 64 Determination of natural diet...... 64 Preference trials...... 65 DISCUSSION...... 66

ACKOWLEDGEMENTS...... 70

REFERENCES...... 71

CHAPTER FOUR RARE CAPTURE OF HYDROTHERMAL VENT-ASSOCIATED OCTOPUS REVEALS SURPRISING INFORMATION ABOUT DIETARY HISTORY...... 85

ABSTRACT...... 86

INTRODUCTION...... 87

METHODS...... 90

RESULTS...... 92

DISCUSSION...... 93

REFERENCES...... 99

viii LIST OF TABLES

Table 1. Summary of literature records of a significant reduction in seastar predation on scallops with sponge encrusted valves over scallops without sponges (“yes” means reduction was demonstrated, no means it was not). Additionally, the portion of diet consisting of of scallops for each seastar species obtained from in situ observations is included (Mauzey et al., 1968)...... 77

Table 2. Number of clean and sponge-encrusted scallops consumed by four Enteroctopus dofleini and six Octopus rubescens during prey choice trials. Choices were significantly different for each prey type in both E. dofleini (Quade test, Quade F = 15, num df = 1, denom df = 3, P = 0.03047) and O. rubescens (Quade test, Quade F = 30.625, num df = 1, denom df = 5, P = 0.002643). Drill holes on scallops consumed by E. dofleini differed significantly from a random distribution (χ2 with Yates's correction= 11.572, df = 2, P = 0.003), with a greater than expected number of drill holes in the encrusted scallops...... 78

Table 3. Summary of known associations between Chlamys genus scallops and sponges...... 79

Supplemental Table 1: Counts of prey (minimum number of individuals) recovered from eight Enteroctopus dofleini middens (ED1-8) and fifteen Octopus rubescens middens (OR1-15) ...... 83

Supplemental Table 2. Literature stable isotopic values from vent and abyssal fauna used for comparison to Graneledone eye lens...... 109

ix LIST OF FIGURES

Figure 1: A: Relationship between the diameter of the eye lenses of seven Humboldt squid (Dosidicus gigas) and their wet mass as successive outer layers are removed (grey open circles) and six whole dried eye lenses (black closed circles). B: Relationship of the dry mass composition of the eye lens of a Humboldt squid and the position within the lens (0% is the center of the lens, 100% is the outermost margin). The relationship is described by the natural log function shown in graph...... 43

Figure 2: Hierarchical clustering dendrograms (average-linkage agglomerative, euclidean distance) of outer 8.4 mm of eye lens stable isotope values of Dosidicus gigas. Tips are shaded according to collection location. LOESS regression track of eye lens stable isotope values are shown below tips with values included in the analysis shown in black, those excluded shown in grey...... 44

Figure 3: Comparison of isotope value of natal Dosidicus gigas eye lens layers to published values. Vertical gray lines are ranges of published values of muscle isotopic values by latitude, while the black solid line is the LOESS regression of those values. Horizontal broken lines are mean isotopic values of natal eye lens (2 mm) for squid in this study grouped by collection location. Nitrogen isotopic values corrected to equivalent muscle values (+2.03 ‰). Histograms represent distribution of all stable isotope values for all lens layers for all squid collected in northern latitudes in this study (squid collected in the Sea of Cortez are excluded)...... 45

Supplementary Figure 1: Example of sequence of nitrogen stable isotope values in right (black) and left (white) eye lens layers of Dosidicus gigas. Line is LOESS smoothed trendline (span=0.3)...... 46

Supplementary Figure 2: Eye lens layer carbon stable isotope patterns (LOESS smoothed) for all Dosidicus gigas used in this study...... 47

Supplementary Figure 3: Eye lens layer nitrogen stable isotope patterns (LOESS smoothed) for all Dosidicus gigas used in this study...... 48

Supplementary Figure 4: Carbon and nitrogen stable isotope values for sequential eye lens layers of individual Dosidicus gigas (Grey circles) compared to most common prey items (Black circles, mean +- SE). Grey circles are proportional in diameter to the diameter of the eye lens at each layer. Dosidicus gigas isotope composition values have been corrected for trophic enrichment (-3.3‰ δ15N) and for difference from muscle (+2.03‰ δ15N). Values for Euphasia pacifica (EUPA), Thysanoessa spinifera (THSP), Clupea pallasi (CLPA), Merluccius productus (MEPR), Engraulis mordax (ENMO), Diaphus theta (DITH), Gonatus onyx (GOON), Stenobrachius leucopsarus (STLE), and Tarletonbeania crenularis (TACR) taken from literature...... 49

x Supplementary Figure 5: Carbon and nitrogen stable isotope values for sequential eye lens layers of individual Dosidicus gigas (Grey circles) compared to most common prey items (Black circles, mean +- SE). Grey circles are proportional in diameter to the diameter of the eye lens at each layer. Dosidicus gigas isotope composition values have been corrected for trophic enrichment (-3.3‰ δ15N) and for difference from muscle (+2.03‰ δ15N). Values for Euphasia pacifica (EUPA), Thysanoessa spinifera (THSP), Clupea pallasi (CLPA), Merluccius productus (MEPR), Engraulis mordax (ENMO), Diaphus theta (DITH), Gonatus onyx (GOON), Stenobrachius leucopsarus (STLE), and Tarletonbeania crenularis (TACR) taken from literature...... 50

Supplementary Figure 6: Carbon and nitrogen stable isotope values for sequential eye lens layers of individual Dosidicus gigas (Grey circles) compared to most common prey items (Black circles, mean +- SE). Grey circles are proportional in diameter to the diameter of the eye lens at each layer. Dosidicus gigas isotope composition values have been corrected for trophic enrichment (-3.3‰ δ15N) and for difference from muscle (+2.03‰ δ15N). Values for Euphasia pacifica (EUPA), Thysanoessa spinifera (THSP), Clupea pallasi (CLPA), Merluccius productus (MEPR), Engraulis mordax (ENMO), Diaphus theta (DITH), Gonatus onyx (GOON), Stenobrachius leucopsarus (STLE), and Tarletonbeania crenularis (TACR) taken from literature...... 51

Figure 4: Locations of collection sites for Enteroctopus dofleini, Octopus rubescens and octopus prey items. Salish Sea has been denoted by light grey in insert, all other marine waters in dark grey...... 80

Figure 5: Treemap of midden contents of 15 Octopus rubescens middens and 8 Enteroctopus dofleini middens. Relative number of each prey species found in all middens is represented by the area of the box, while the shading of the box represents the proportion of middens in which prey species was observed. Each prey species is then situated hierarchically into higher taxonomic groups so that the relative representation of these higher groups in the middens can be easily compared. Prey species composing less than 5% of midden items are lumped into Other categories within each high taxonomic category. Plot was generated using the tmPlot function from the ‘treemap’ package for R (R Development Core Team, 2008). For prey item counts for all prey species recovered from each den see Table S1...... 81

Figure 6: LOESS smoothed nitrogen and carbon stable isotope composition series of the eye lens layers of three Enteroctopus dofleini (right) collected in the Salish Sea. E. dofleini δ15N values were corrected for trophic enrichment by subtracting 3.3‰. Diameter of bubble corresponds to the diameter at which eye lens layer was collected, therefore larger bubbles reflect isotope composition from later in life. Common prey items are also plotted by initials (Cp=Cancer productus, Pg=Pugettia gracilis, Bn=Balanus nubilus, Ch=Chlamys hastata, Cr=Chlamys rubida, Hk=Humilaria kennerlyi, Sg=Saxidomus gigantea, Cn=Clinocardium nuttallii, Cf=Calytrea fastigiata, Am=Acmaea mitra, Tt=Terebratalia transversa, Mi=Myxilla incrustans). Three potential prey-item clusters determined by model-based cluster analysis are identified by boxes...... 82

Figure 7: Map of Graneledone collection location in the Dead Dog vent field of Middle Valley on the Juan de Fuca Ridge vent system (left), and geographic location of Middle Valley, and locations of collection points of literature obtained potential prey items (right). Black lines in right map indicate tectonic plate margins...... 106

xi Figure 8: Graneledone cf boreopacifica eye lens layer carbon and nitrogen stable isotope values (gray circles) plotted versus isotopic values of potential prey items from vent (black circles) and abyssal (white circles) food webs. Vent genera that has been found in Graneledone cf boreopacifica guts are marked with a white “x”. Graneledone eye lens value circles have been scaled proportionally to eye lens diameter at a given layer, thus large circles indicate layers for later in life. Graneledone eye lens δ15N values were reduced by 3.3 ‰ to correct for trophic enrichment of 15N. Stable isotope composition of muscle taken from both arm and mantle is shown as dark grey circle and marked (mean ± SD, also reduced by 3.3 ‰ δ15N)...... 107

Figure 9: Proportion composition of hydrothermal vent fauna (black) and abysal fauna (gray) in the lifetime diet of a Graneledone cf boreopacifica collected from Middle Valley, Juan de Fuca Ridge vent system as determined using a bayesian stable isotope mixing model (IsotopeR) from carbon and nitrogen stable isotope compositions of sequential eye lens layers. White dashed line indicates lower 95% confidence interval; upper 95% CI is 1 at all lens diameters and is thus not shown. Figure B shows expanded view of proportions 0.91 to 0.96 to better show trend...... 108

xii Dedication

This work is dedicated to my father, James W. Onthank who dreamed of becoming a marine biologist when he was young but instead created one he would never meet I miss you

And to my wife, Stephanie J. Onthank who never imagined a simple suggestion before we were married would lead to having a part-time husband for the last five years Thank you for your patient endurance

xiii CHAPTER ONE

GENERAL INTRODUCTION

Cephalopods are major components of worldwide marine biomass and occur in nearly every marine habitat (Boyle and Boletzky 1996), and consequently are important members of most marine food webs (Clarke 1996b). Cephalopods are especially important prey of cetaceans and pinnipeds, but also important components in the diets of many marine birds and fishes

(Clarke 1996b). Two cephalopods common in the northeastern Pacific ocean are exemplars of this: a small octopus, the east Pacific red octopus, Octopus rubescens, and a large squid, the

Humboldt squid, Dosidicus gigas. Octopus rubescens is a major component in the diet of harbor seals, Phoca vitulina richardsi, representing the single most common prey species in some regions, composing up to 40% of the diet (Oxman 1995), becoming exceptionally important during El Niño events when other food items become scarce (Stewart and Yochem 1994).

Octopus rubescens is also a component in the diets of California sea lions (Lowry et al. 1990;

Orr et al. 2011), sea birds such as pelagic cormorants and Brandt's cormorants (Ainley et al.

1981), common murres (Ainley et al. 1996), and fishes such as chinook salmon (Hunt et al.

1999), sandpaper skates (Rinewalt et al. 2007) and longnose skates (Robinson et al. 2007).

Dosidicus gigas is a major prey item of sperm whales where the species co-occur and sperm whales appear to adjust their behavior based on the abundance of D. gigas in the local environment (Jaquet and Gendron 2002). Additionally, D. gigas appears to be one of the most commonly preyed upon species by major billfishes in the eastern Pacific including blue marlin

(Abitia-Cardenas et al. 1999), sailfish (Rosas-Alayola et al. 2002), and swordfish (Markaida and

1 Hochberg 2005).

The role of cephalopods as predators has been less well characterized, but this molluscan group likely is important in structuring benthic food webs. Cephalopods have among the highest metabolisms of marine predators, even higher than comparably sized fish (Seibel and Drazen

2007), and therefore likely consume more prey than their biomass may initially suggest.

Octopuses have been suggested to be "switching predators" that vary diet according to prey abundance and help stabilize prey populations (Vincent et al. 1998).

Like many marine fisheries, cephalopods are facing recent increases in threats to their continued productivity. Cephalopod fisheries have risen dramatically over the past 40 years, quadrupling the amount of cephalopod landings (1 to 4 million metric tons annually), and tripling in share of marine fisheries (2% to 6% annually) over that time period (FAO 2013).

Increased global atmospheric CO2 levels have lead to a decrease in marine pH, known as ocean acidification (Doney et al. 2009). Cephalopod hemocyanins are exceedingly sensitive to pH

(large Bohr effect) and drops in environmental pH would reduce oxygen loading in the gills of cephalopods and has been shown to reduce oxygen consumption in Dosidicus gigas (Fabry et al.

2008).

Despite this apparent ecological importance and increasing threats, relatively little is known about cephalopod life histories (Clarke 1996a). This has happened for several reasons: until recently little attention has been paid to the ecological contributions of cephalopods (Clarke

1996a), rearing cephalopods in captivity is difficult and very few species can be hatched and raised to adulthood in aquaria (Iglesias et al. 2007). Attempts to study cephalopods in situ have been hampered by the difficulty of tagging cephalopods long term, especially in octopuses

2 (Semmens et al. 2007), although shorter-term tagging (1-4 weeks) of squid has proven successful

(Sauer et al. 2000; Gilly et al. 2006; Bazzino et al. 2010). As with many marine organisms, it is also difficult in many of these species to determine diet, and thus their predatory role in an ecosystem. Analysis of stomach contents has historically been the method of choice for quantifying cephalopod diets (Smith 2003; Field et al. 2013), but only in those species that are the target of major fisheries are a sufficient number of individuals collected for this to be a reliable method. Also hindering this method is the issue that prey, especially soft bodied taxa, are often difficult to identify after ingestion and partial digestion, and stomach contents only reflect recent feeding, sometimes abnormal feeding that occurred during the collection process such as feeding in trawl nets and capture induced cannibalism as seen in D. gigas (Ibáñez and

Keyl 2010).

This dissertation explores the potential of using stable isotope analysis (SIA) of archival tissues to expand our knowledge of cephalopod movements and diets. Stable isotope ratios, particularly the ratios of carbon-13:carbon-12 and nitrogen-15:nitrogen-14 have proven useful to help determine diets of marine organisms (Michener and Kaufman 2007) and to elucidate movement patterns (Hobson 1999; Hobson 2007). Heterotrophic organisms obtain virtually all of the nitrogen and carbon in their tissues from their diet, so tissue stable isotope composition is related to dietary stable isotope composition. The bulk carbon isotope composition of an organism generally reflects dietary carbon isotope composition. An organism's nitrogen stable isotope composition, however, will be approximately 3‰ δ15N greater than dietary composition, a phenomenon known as trophic enrichment (Michener and Kaufman 2007). There are also large-scale geographic patterns in relative stable isotope abundance, commonly know as

3 isoscapes (Bowen, 2010), that can be leveraged to track animal movement through these areas

(MacKenzie et al, 2011). Archival tissues are those with no elemental turnover after formation, so stable isotope composition in these tissues is related to diet at the time of formation.

Stable isotope analysis of archival tissues has been utilized in highly mobile marine organisms to elucidate movement patterns without the need of tagging animals. Archival tissues that have helped reveal dietary or geographic changes include whiskers in fur seals (Cherel et al.

2009), dentin in teeth in sperm whales (Mendes et al. 2007), bottlenose dolphins (Knoff et al.

2008), orcas (Newsome et al. 2009), Steller's sea lions (Hobson and Sease 1998), and southern elephant seals (Martin et al. 2011), baleen in bowhead whales (Hobson and Schell 1998; Lee et al. 2005) and right whales (Best and Schell 1996), the otoliths of snapper (McMahon et al. 2011), salmon (Wurster et al. 2005), orange roughy (Shephard et al. 2007), atlantic cod (Schwarcz et al.

1998) and many other fish species, and the vertebrae of white sharks (Estrada et al. 2006). The use of stable isotope analysis of the archival tissues in cephalopods could help ameliorate the lack of life history knowledge in cephalopods. However this class of mollusks contains very few hard tissues, and none of the aforementioned. An archival tissue is, at a minimum, metabolically inactive, but is ideally also a tissue that is formed continuously throughout the life of the organism, old portions are not eliminated from the organism, and can be separated into subsets that are formed at temporally distinct intervals. There are four potentially useful archival tissues in cephalopods that were considered for the studies in this dissertation: beak, gladius, statolith and eye lens.

The use of cephalopod beaks has been explored by several studies (Cherel and Hobson

2005; Ruiz-Cooley et al. 2006; Hobson and Cherel 2006), however there is not a good model for

4 cephalopod beak growth, so separating new from old beak material is problematic. Additionally cephalopod beaks exhibit large gradients of protein, chitin, and pigmentation across the beak which will likely skew stable isotope compositions (Miserez et al. 2008).

The gladius (pen) in squid is an archival tissue from which investigators have obtained sequential records of stable isotope composition by sectioning the length of the proostracum

(Ruiz-Cooley et al. 2010; Lorrain et al. 2011). The gladius bears growth lines and has been long used to age and determine growth curves of individual squid (Arkhipkin and Perez 1998).

Additionally gladius length is very closely related to mantle length (the gladius generally determines the length of mantle), the primary measurement used to size squid (Perez et al. 2006).

The gladius, however, does not only grow in length, but also grows in thickness over the lifetime of the squid as lamellae are added to the ventral surface, meaning that sectioning the gladius by length does not yield temporally distinct samples (Arkhipkin and Perez 1998). Additionally that gladius is missing in most other cephalopod groups, such as octopuses, cuttlefish, and sepiolids, giving this structure limited utility across the class.

Statoliths are calcareous elements used by cephalopods to determine the direction of gravity and have been used extensively to determine age in cephalopods, as they often exhibit prominent growth rings (Bettencourt and Guerra 2000). These structures, however, have not been explored as a potential archival tissue for stable isotope analysis, and only sparsely for trace element analysis as otoliths have been used in fish (Ikeda et al. 2002; Ikeda et al. 2003).

Cephalopod statoliths are much smaller than fish otoliths. Large squid, such as adult Dosidicus gigas, bear statoliths as small as 2-3mm (pers obs). At this size the statoliths are hard to sample and do not provide enough material for sectioning and analysis in traditional stable isotope mass

5 spectrometers, especially in small species.

Eye lenses have been used as an archival tissue for stable isotope analysis in only a single published study (Hunsicker et al. 2010) and one unpublished portion of a doctoral dissertation

(Parry 2003). In vertebrates the bulk of the lens is composed of fibers which lack nuclei and mitochondria and have essentially no metabolic activity. The lens epithelium is metabolically active, and lies on the anterior portion of the lens, where it deposits layers of fibers (Berman

1991). Similarly the lenses of cephalopods are produced by the metabolically active lentigenic cells exterior to the lens, but the lens itself is devoid of nuclei (West et al. 1995), and is likely metabolically inactive. Cephalopod eye lenses are composed of many concentric layers (West et al. 1995), presumably generated in a daily fashion (Richard Young, pers comm). Fish eye lenses have demonstrated utility in differentiating populations due to differential trace metal incorporation and have a high degree of agreement with otoliths (Dove and Kingsford 1998). The concentric layers of cephalopod eye lenses can easily be separated by peeling layers of lenses apart that have not yet been dried. This yields subsections that are temporally distinct.

Additionally, eye lenses are of a size to be easily manipulated and provide sufficient material for traditional stable isotope mass spectrometry. In this dissertation I demonstrate the utility of stable isotope analysis of cephalopod eye lenses by using this approach to address three questions in cephalopod biology.

All subsequent chapters include coauthors, including Dr. Raymond Lee, who is my major professor and in whose lab most work was conducted. I, however, am the primary author and intellectual originator of all work found in this dissertation.

In the second chapter of this dissertation I use SIA of eye lenses to address movement

6 patterns in Dosidicus gigas, a large squid that is currently extending its range northward in the northeast Pacific ocean. This manuscript has been prepared for submission to the Canadian

Journal of Fisheries and Aquatic Sciences with Dr. Ray Lee, my committee chair in whose lab I work included as coauthor.

The third chapter addresses the possibility that octopus predation may play a role in maintaining a symbiosis between scallops and sponges in the Salish Sea. Here SIA of eye lens layers enabled me to verify dietary information from midden analysis and also identify the sizes of octopuses most likely to be scallop predators. This manuscript has been prepared for and submitted to the Journal of Molluscan Studies. In addition to Ray Lee, my major professor, Dr.

David Cowles from Walla Walla University is a coauthor on this paper as I used his lab space at

Rosario Beach Marine Laboratory (RBML) to conduct much of the work.. Several RBML students who helped in sample and data collection were included as co authors of this manuscript as well. Specifically, Brianna Payne and Ashley Groeneweg helped conduct prey preference trials on three Enteroctopus dofleini; Thomas Ewing and Nicolas Marsh help collect prey remain from middens of Enteroctopus dofleini. All authors reviewed and commented on the manuscript.

Finally, the fourth chapter utilizes SIA of eye lens of a single deep-sea, hydrothermal vent associated octopus, Graneledone cf boreopacifica, to determine relative contributions of vent and non-vent fauna in the diet. The evidence presented in this chapter demonstrates that this individual surprisingly consumed little or no vent fauna, despite being collected a few meters from chemosynthetic communities, and represents a considerable expansion in the knowledge of the diet of vent-associated Graneledone. This manuscript has been prepared for submission to

7 Marine Biology with Dr. Ray Lee, my committee chair in whose lab I work included as coauthor.

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17 CHAPTER TWO

CARBON AND NITROGEN STABLE ISOTOPE ANALYSIS OF EYE

LENSES SUGGEST NATAL ORIGINS AND MIGRATORY HISTORIES OF

NORTHWARD RANGE EXPANDING HUMBOLDT SQUID (DOSIDICUS

GIGAS)

Kirt L. Onthank* and Raymond W. Lee

School of Biological Sciences, Washington State University

PO Box 644236, Pullman, WA 99164-4236

*-corresponding author

Email: [email protected]

Formatted for: Canadian Journal of Fisheries and Aquatic Sciences

18 ABSTRACT Over the last decade the Humboldt squid, Dosidicus gigas, has expanded the northern boundary of its historical range in the eastern Pacific, periodically being found as far north as

Canada and Alaska. There is a paucity of data available to elucidate the origin or migration history of these squid arriving in northern latitudes. In this study we attempt to address this problem by using carbon and nitrogen stable isotope analysis of eye lenses, an archival tissue, to provide evidence of the histories of individual squid. We found the squid had highly variable carbon and nitrogen stable isotope records, suggesting highly divergent migration histories even between squid collected in close proximity. Additionally natal eye lens layers of all squid collected in US and Canadian waters showed exceptionally low δ13C values, indicating an origin north of 37° N.

Keywords: Dosidicus gigas, stable isotopes

19 INTRODUCTION Recent years have seen a northward range expansion of the large ocean squid, Dosidicus gigas, in the eastern Pacific (Zeidberg and Robison 2007). Historically D. gigas has ranged as far north as the coastal waters of the Baja California peninsula. However, since the 1997/98 El

Niño event this species has been regularly encountered in coastal California, and occasionally in large numbers as far north as British Columbia with individuals found as far north as Alaska

(Cosgrove and Sendall 2005). It has been proposed that these D. gigas are migrating northward during warm months from historical habitats to take advantage of high productivity of coastal US and Canadian waters before returning to southern habitats in the fall (Field et al. 2013).

Cephalopods, such as D. gigas, are a vital component of oceanic ecosystems serving not only as important predators but also prey items, particularly of many apex predators (Rodhouse and Nigmatullin 1996; Abitia-Cardenas et al. 1999; Rosas-Alayola et al. 2002; Markaida and

Hochberg 2005). As such, changes to the distribution of major cephalopod populations would likely have a dramatic effect on the ecosystems vacated or colonized. This may include impacts on commercially important fish stocks such as Pacific hake (Merluccius productus), Pacific sardine (Sardinops sagax), and Pacific herring (Clupea pallasii), which have been shown to compose a sizable portion of D. gigas diets in northern latitudes (Braid et al. 2012; Field et al.

2013).

Hypotheses of the cause of this northward expansion have included impacts of fisheries on squid predators in invaded regions, shifts in water temperature following strong El Niño/La

Niña, and geographic and vertical expansion of the oxygen minimum zone in the northeastern

Pacific (Zeidberg and Robison 2007; Keyl et al. 2008; Stewart et al. 2013a). The search for a

20 driver of the movement of D. gigas into northern latitudes, however, is hampered by the stark lack of any data concerning the origins of the squid arriving in coastal US and Canadian waters and the possible routes taken from this hitherto unknown origin. Here we attempt to address this paucity of data concerning the northward migration of these squid by using stable isotope analysis of archival tissues of northward migrant squid.

Stable isotope analysis is an emerging tool that has been used to elucidate movements and migratory origins of wide-ranging marine organisms, including determining migratory origins of loggerhead turtles (Caretta caretta) in the Southern California Bight (Allen et al. 2013). Recently independent investigations have provided extensive data on variation of carbon and nitrogen stable isotopes in D. gigas over the entirety of this species' historical and expanded range (Ruiz-

Cooley and Gerrodette 2012; Argüelles et al. 2012). Modeling this variation in δ13C and δ15N values in mantle muscle of D. gigas have explained 77% and 82% of observed variation, respectively (Argüelles et al. 2012). Approximately 90% of the explained variation in δ13C values was due to factors relating to the location of the squid such a latitude and distance from continental shelf break, while 63% of the explained variation in δ15N values was due to location factors. In another investigation latitude was also found to be the primary factor in variation in

δ13C and δ15N values, and the patterns in variation remained largely unchanged when considering secondary variables such as squid size, distance offshore, and year of collection (Ruiz-Cooley and Gerrodette 2012). This work has provided a valuable baseline to compare future D. gigas investigations utilizing carbon and nitrogen stable isotopes.

Particularly useful in such investigations is the use of archival tissues that are largely metabolically inert and have little elemental turnover after formation. Such tissues provide a

21 record of past isotopic composition of an organism. Archival tissues such as the baleen of mysticete whales, teeth in sperm whales, and barnacle plates found on loggerhead turtles have been used to infer migration history of large, wide-ranging marine organisms (Best and Schell

1996; Lee et al. 2005; Mendes et al. 2007; Hobson 2007). Two potential archival tissues in squid, the gladius and eye lens, have each been used to infer past isotopic signatures in squid.

Investigations exploring the use of the gladius have been able to differentiate between D. gigas collected at differing locations and showed little evidence of migration between locations (Ruiz-

Cooley et al. 2010). Other investigations using D. gigas gladius have shown that individual squid can have highly divergent dietary and movement patterns (Lorrain et al. 2011). Use of eye lenses on a population level in Ommastrephes bartramii, confamilial with D.gigas, have revealed that the sequence of stable isotope composition in individual squid eye lens layers closely corresponds to the sequence of stable isotope composition of mantle muscle of multiple individuals of increasing size in the population, strongly suggesting that the eye lens layers record the isotopic history of these animals (Parry 2003). Stable isotope analysis of the eye lenses of Berryteuthis magister have been used to demonstrate temporal variations in carbon and nitrogen stable isotope composition too fine to be reflected in mantle muscle (Hunsicker et al.

2010).

In this study we bring carbon and nitrogen stable isotope analysis of archival tissues to bear on the question of origin and migratory history of the D. gigas which have recently expanded their range northward in the eastern Pacific.

22 MATERIALS AND METHODS Eye lenses of Humboldt squid, Dosidicus gigas, were collected from the United States west coast between October 2008 and June 2010. Five squid were collected near Tofino, British

Columbia, Canada ( 49° 3' 23.32"N 125° 43' 39.04"W, size range: 53-61 cm ML), eleven squid were caught by a Pacific hake fishing vessel at 494 m depth off the coast near Astoria, OR (46°

08' 18” N 124° 44' 44” W, size range: 60-72 cm mantle length (ML)); one was collected alive from the marina dock in Westport, WA (46° 57' 27” N 124° 06' 30” W , size 59 cm ML), and eleven were collected in California, (35° 42' 12” N 121° 52' 0” W, size range 39-69 cm ML) approximately 189 km northwest of Point Conception at a depth of 320 m, five were collected in the Sea of Cortez, Mexico (27° 34' 27” N 111° 43' 27” W) approximately 21 km northwest of

Isla Tortuga. Heads and internal organs were collected (OR and WA specimens) or only eye lenses (BC, CA, and MX squid). All squid tissues were frozen and shipped to Pullman, WA for isotopic analysis.

Squid from Oregon and Washington were thawed and eye lenses were immediately dissected out. Lenses removed from the animals were immediately sectioned if possible, or stored frozen in a -20 °C freezer until sectioning was possible. Cephalopod lenses form in two sections, anterior and posterior. The anterior lens was removed and discarded. The posterior lens, which is much larger, was sectioned for analysis. Sectioning involved peeling successive growth layers groups (GLG) from the outer portion of the lens under a stereoscopic dissecting microscope. Before the removal of a GLG, the lens was weighed and the diameter measured.

Excised GLGs were placed into separate microcentrifuge tubes and dried at 60 °C for at least 24 hours. In this manner we were able to recover an average of 40 roughly equally sized GLGs per eye lens of adult Humboldt squid. Since this species of squid is thought to live approximately

23 one year (Nigmatullin et al. 2001), this sampling should provide less than 10-day temporal resolution. The GLG position in the lens was recorded as the diameter (mm) of the remaining lens before the GLG was removed. For instance, in a lens with a total diameter of 15 mm, the first GLG removed would be 15 mm. Each successive layer removed would correspond to the smaller diameter of the remaining lens, approaching zero toward the center. Smaller GLG sample diameters are from earlier in the life of the animal. Additionally, muscle tissue was collected from the arms of seven squid collected in Oregon to compare to eye lens tissue. Lipids were not extracted from muscle tissue before isotopic analysis, however a +0.8 ‰ δ13C and +0.68 ‰ δ15N correction was applied to measured values to correct to lipid extracted equivalents (Ruiz-Cooley et al. 2011).

Dried samples were ground to a fine powder and approximately 0.50 mg was placed into a tin capsule for isotopic analysis of 13C/12C and 15N/14N ratios. Samples were analyzed using a

Eurovector elemental analyzer in line with a Micromass Isoprime continuous flow stable isotope mass spectrometer. Results are presented in the standard δ notation, where isotopic ratios are expressed in ‰ differences relative to the conventional standard, the PeeDee Belemnite limestone formation for carbon and atmospheric nitrogen. Where:

R δ = sample −1 ×1000  Rstandard 

Routine precision for δ13C and δ15N was ± 0.1 ‰ and 0.3 ‰ respectively.

Lenses were sectioned undried because dried lenses became brittle and difficult to section. This, however, presented problems in comparing lenses in different sized individuals due to a water content gradient along the diameter of the lens. In order to compare lenses of

24 individuals of different sizes, lens diameters and weights were corrected to dry equivalents. The water content gradient of D. gigas lenses was empirically determined by sectioning two lenses and weighing layers before and after drying.

To estimate mantle length from eye lens diameter a linear regression of dry mass corrected diameter compared to mantle length of squid collected in Oregon and California was performed with y-intercept specified as 0. To compare isotope values for D. gigas eye lenses hierarchical clustering was employed. LOESS regression with a span of 0.3 was applied to each series of lens layers. Values for this regression were predicted at 0.3 mm (the average sampling frequency) for the outer 8.4 mm of each lens (final layers formed common all squid) were then used for average linkage agglomerative hierarchical clustering analysis.

A paired t-test was performed to compare lipid extraction corrected muscle tissue δ13C and δ15N values and outermost (most recent) eye lens layers. If a significant difference was found between muscle and eye lens layers, the mean difference was applied as a correction to eye lens layers for comparisons to muscle tissue.

All statistical analyses and regressions were performed using R (R Core Development

Team, 2012). For comparison to eye lens layers occurring in the innermost 2mm of eye lenses

(natal lens layers, corresponding to a estimated mantle length of 0.085m) of squid collected in this study, δ13C and δ15N values for D gigas were collected from literature sources (Ruiz-Cooley et al. 2006; Ruiz-Cooley et al. 2010; Lorrain et al. 2011; Ruiz-Cooley and Gerrodette 2012;

Argüelles et al. 2012), including using g3data software to extract data from graphs (Bauer and

Reynolds 2008). Stable isotope compositions of the most important prey items of northern D. gi gas (Field et al. 2013) were taken from literature sources and were collected from coastal west-

25 ern United States between 41.9N and 46.3N (Bosley et al. 2004; Miller et al. 2010) except the myctophid fish Tarletonbeania crenularis, which was collected in the Gulf of Alaska between

58N and 59N (Kline Jr 2010). Stable Isotope composition values for this species was corrected using shifts in values for other myctophid species collected at both locations.

RESULTS Percent dry mass of D. gigas eye lenses decreased in a logarithmic fashion from the center to exterior of the lens (Fig 1). By integrating the natural log relationship in Figure 1-B between moisture and lens layer position such that it instead relates mass remaining interior to a given layers' dry mass to wet mass yields:

D M = M ⋅ 0 .846 − 0.066⋅ln 1.345− sample dry wet D [  total ]

Where Mdry the dry mass of the eye lens including and interior to a specific sampled layer, Mwet is the wet mass of the lens including and interior to a specific sampled layer, Dsample is the diameter of the lens at the sampled layer and Dtotal is the total diameter of the entire lens. Further, combining this with the relationship between mass and diameter resulted in the following function that describes the relationship between the diameter at which a lens layer lies in dried versus wet lens.

0 .331 D D =D ⋅ 0.846 − 0.066 ⋅ln 1.345− wet dry wet D [  total ]

26 Where Ddry is the diameter of the dried lens at a specific sampled layer, Dwet is the diameter of the undried lens at a specific sampled layer, and Dtotal is the diameter of the whole undried lens. All lens diameters were corrected to dry diameters using this equation.

Lipid extraction corrected muscle and eye lens tissue showed significant differences in

δ15N values (Paired t-test, t=14.5611, df=6, p<0.0001), but not in δ13C (Paired t-test, t=1.6763, df=6, p=0.1447). Muscle tissue had a mean δ15N value of 2.03 ‰ (± 0.37 sd) greater than eye lens values, and will be used to correct eye lens δ15N values when comparing to muscle δ15N values. This closely agrees with previous investigations using stable isotope analysis of squid eye lenses, which have found outermost eye lens layers to be relatively depleted in 15N compared to muscle tissue, but did not report a difference in carbon isotope values (Parry 2003; Hunsicker et al. 2010). The difference between eye lens and raw muscle δ15N values found in this study (1.2

‰ in D. gigas) was similar to previous studies (1.0 ‰ in O. bartramii and 1.1 ‰ in S. oualaniensis, Parry 2003).

Stable isotope values were very consistent between left and right eye lenses (Supp Fig 1).

Stable isotope composition of squid eye lenses collected in this study fit within the range expected for squid feeding in northern latitudes on the most important prey items of Dosidicus gi gas (Supp Figs 4-6). There is no apparent common ontogenetic trend in stable isotope compositions of eye lenses among squid. Individual squid, however, do exhibit changes in stable isotope composition in eye lenses suggesting shifts in diet over the lifetime of the squid. These shifts are particularly evident in nitrogen stable isotope composition. Squid eye lens did not cluster by geographic location of collection by either δ13C or δ15N values (Fig 2).

27 Mean δ13C values from natal eye lens layers (California = -18.6 ± 0.7 ‰, Oregon/Wash- ington = -18.4 ± 0.5 ‰, British Columbia = -18.2 ± 0.3 ‰) were lower than the majority of values found in literature reports for D. gigas (Fig 3). The mean natal eye lens δ13C values of squid collected in British Columbia and Oregon only overlapped literature values for squid collected between the latitudes 1.6°-6.8° S and 37.6°-46.1° N. δ13C values for squid collected from Cali- fornia were even lower and more restricted in the latitudes matched: 1.6°-5.3° S and 38.7°-46.1°

N. Natal eye lens layer δ13C values for smaller squid collected from the Sea of Cortez, Mexico were considerably higher than those from squid collected farther north. Natal eye lens layer corrected δ15N values of squid in this study (California = 13.5 ± 1.0 ‰, Oregon/Washington = 12.8 ±

0.8 ‰, British Columbia = 14.1 ± 1.6 ‰) were more comparable to literature values than δ13C, with the only latitudes at which literature reported values appear well outside those reported here is a narrow band of latitudes for which exceptionally low δ15N values have been reported for D. gigas centering on approximately 5° S.

DISCUSSION

Diet In this study we have employed stable isotope analysis of an archival tissue, eye lenses, in

D. gigas to explore migratory history of individuals from their expanded northern range. Pat- terns of eye lens δ15N values did not show a consistent increase with increasing squid size (Supp

Figs 2-3). The lack of an ontogenetic increase in δ15N values of eye lens values with size in individuals is somewhat surprising. Most aquatic predators eat prey from higher trophic levels as the organism grows, resulting in tissue δ15N values that rise over the course of the predators' lifetime

(Cocheret de la Morinière et al. 2003; Estrada et al. 2006). This increase of δ15N values in mus-

28 cle tissue with increasing squid size has been demonstrated in Ommatrephes bartrammi, Stheno teuthis oualaniensis, and Berryteuthis magister, however when sequential eye lens δ15N values are observed ontogenetic shifts in δ15N of individuals appears to be much more complicated, but in general recapitulate general trends seen at the population level (Parry 2003; Hunsicker et al.

2010). Similarly, in Dosidicus gigas, when muscle nitrogen stable isotope values are compared between individuals of varying size there is an apparent increase in δ15N values with size (Ruiz-

Cooley et al. 2006), however when individual ontogenetic changes are examined using archival tissue regular increases in δ15N with size are not as apparent, with many squid showing a strong decrease in δ15N with increasing size (Ruiz-Cooley et al. 2010; Lorrain et al. 2011).

The squid in this study appear to shift at various times in life between consuming prey items at two tropic levels, one containing mostly euphausiids (krill: Euphausia pacifica &

Thysanoessa spinifera) and one trophic level higher containing small fish including myctophids

(Tarletonbeania crenularis, Stenobrachius leucopsarus,and Diaphus theta), northern anchovie

(Engraulis mordax), Pacific hake (Merluccius productus), and Pacfic herring (Clupea pallasi)

(Supp Figs 4-6). This finding reinforces the importance of euphausiids in the diet of Dosidicus gigas at northern latitudes. A previous analysis of D. gigas stomach contents found that only fish of the family Myctophidae occuring more frequently than crustaceans of the family Euphausiidae

(Field et al. 2013). This also confirms previous studies that have failed to show a consistent increase in D. gigas trophic level with size (Ruiz-Cooley et al. 2010; Lorrain et al. 2011; Ruiz-

Cooley and Gerrodette 2012) and provides additional evidence highlighting opportunisitic foraging behavior in D. gigas, including foraging on prey as small as euphausiids into adulthood

(Ibáñez et al. 2008; Field et al. 2013).

29 There is strong evidence that shifts in eye lens stable isotope composition reflect changes in dietary isotope composition. Previous investigations have found that stable isotope composition sequences in the eye lenses of multiple individuals closely follows stable isotope composition of mantle muscle by size of squid in the same population (Parry 2003). Additionally eye lens isotope compositions observed in this study are consistent with major prey items found in the region, and outermost lens layers are consistent with muscle isotope compositions reported in the literature for D. gigas collected in this region (14.3 ± 1.7 ‰ δ15N , -18.5 ± 0.8 ‰ δ13C)

It is possible for metabolic factors to influence stable isotope composition in a way somewhat independently of dietary sources. Fasting animals have been shown to have muscle δ15N values higher than those seen in those same organisms before the beginning of fasting (Hobson et al. 1993; Cherel et al. 2005). This, however, should not influence the isotopic composition of archival tissues that do not experience elemental turnover after formation. The relative enrichment of 15N in muscle tissue compared to outermost eye lens is consistent with other squid (Parry

2003; Hunsicker et al. 2010), and δ15N values of eye lens seen here are not abnormal for the region or suspected prey items. Nutritional state can also influence δ13C values in tissues, but effects have been varied (Williams et al. 2007; McCue and Pollock 2008), or finding no observed effect of nutritional status on δ13C values. Most of these effects have been attributed to reallocation and catabolism of 13C depleted lipids. Cephalopods, however, assimilate a relatively low percentage of lipids from their diet (Onthank and Cowles 2011), and do not appear to use lipids as a major energy storage medium as many vertebrates do (Lee 1994), so reallocation of lipids is not likely to skew stable isotope compositions of squid considerably.

30 Squid eye lens layers also show a gradient of protein concentration from interior to exterior (Sweeney et al. 2007), which could potentially skew stable isotope ratios. However, studies to date examining carbon and nitrogen stable isotope progression through eye lenses have shown variable trends (Parry 2003; Hunsicker et al. 2010), making a systematic bias caused by the protein gradient unlikely. Additionally, shifts in eye lens layers isotope compositions have reflected shifts in bulk isotope compositions with size in the same population (Parry 2003; Hunsicker et al.

2010), which would be unlikely if eye lens layer stable isotope compositions were skewed based on position in the lens.

Migration The most important factor influencing carbon stable isotope composition of D. gigas is geographic location, with the most important geographic parameter being latitude (Argüelles et al. 2012; Ruiz-Cooley and Gerrodette 2012). In our study the patterns of stable isotope composition of eye lens layers of squid collected in close proximity were not necessarily more similar to each other than to squid collected at more distant locations, suggesting a diversity of long-term migration histories among the squid sampled. This is consistent with shorter-term tagging data that have shown D. gigas individuals do not to move in coherent groups on time scales of days to weeks (Gilly et al. 2006; Bazzino et al. 2010; Stewart et al. 2012; Stewart et al. 2013b). Our data provides additional evidence that suggests that D. gigas is unlikely to form coherent groups and adult squid may in fact have migration histories more similar to squid located over 1500 km away than to squid in the same shoal.

Because there is strong trophic fractionation of nitrogen stable isotopes it is possible for squid residing in the same area, at the same time, but eat prey from different trophic levels to

31 have differing δ15N values. The results of this study show that squid from the same shoal may be eating differing prey items (Supp Figs 4-6). Trophic fractionation, however, is very weak in carbon stable isotopes, and has been shown to be virtually 0 in cephalopods that have been examined (Hobson and Cherel 2006). Therefore carbon stable isotopes would very likely be similar for squid residing in the same area. This has been born out in previous investigations of squid stable isotope composition with δ13C values routinely showing less variability in squid collected from one location than δ15N values in those same squid (Takai et al. 2000; Cherel et al. 2009;

Hunsicker et al. 2010). However, previous surveys of D. gigas muscle isotope composition have shown little variation with latitude north of 37°N in either nitrogen or carbon stable isotope values (Ruiz-Cooley 2012). It would be expected that squid residing long term in this region would not possess isotope compositions that would differentiate based on geography, such as those observed in this study (Fig 2).

δ13C values from all lens layers of all squid match those that would be expected for squid residing lifelong in latitudes north of 37ºN (Fig 2). Literature δ13C values of Humboldt squid muscle from these latitudes range from -19.7 ‰ to -16.7 ‰ (Ruiz-Cooley et al. 2012), while in this study 95% of all eye lens layers ranged between -19.5 ‰ to -17.0 ‰ with a mean of -18.3 ±

0.8 ‰. The range of observed δ15N values, however, slightly exceeded the literature range for northern latitudes of 12.6 ‰ to 16.5 ‰ (Ruiz-Cooley et al. 2012), with 95% of all lens δ15N values in this study falling between 12.5 ‰ to 17.6 ‰, with a mean of 14.9 ± 1.5 ‰. Despite being somewhat higher than literature values, the δ15N values of eye lens layers of nearly all squid in this study comport with values that would be expected for squid feeding on common prey species in these northern latitudes (Supp Figs 4-6). Additionally small squid collected further

32 south in Mexican waters (27.6°N) showed higher δ13C and δ15N values than squid collected at any of the northern localities (Fig 2).

Carbon and nitrogen isotopic composition of innermost eye lens segments can potentially hold information concerning the natal origins of the squid from which it was collected. Natal eye lens layers of D. gigas collected in California, Oregon and British Columbia has exceptionally low δ13C values compared to values that have been previously reported for this species (Ruiz-

Cooley et al. 2010; Lorrain et al. 2011; Ruiz-Cooley and Gerrodette 2012; Argüelles et al. 2012), comparable only to squid collected between the latitudes 1.6°-6.8° S, a southern possible origin

(SPO), and 37.6°-46.1° N, a northern possible origin (NPO). Published δ13C values of squid collected along the Baja California peninsula, often speculated to be the origin of squid occasionally occurring in United States waters (Field et al 2013), do not overlap the mean δ13C value of natal eye lens segments from this study. However, δ15N values of natal eye lens layers in this study correspond well to published values for D. gigas throughout much of the range.

There are, however, challenges for a hypothesis that D. gigas collected in the coastal waters of the US and Canada migrated from either the SPO or NPO. Evidence suggests that D. gi gas eggs do not hatch in waters where the temperature at the pycnocline is cooler than 15°C, which is true of nearshore habitats at the latitudes of the NPO (Staaf et al. 2011). Staaf and col- leagues, however, did identify a region a short distance (~500 km) offshore reaching as far north as 43°N in the months of July-October. Paralarvae of D. gigas have never been observed in waters as far north as the NPO, however observational effort has been very minimal. Squid migrating to coastal US from the NPO could conceivably have varied migratory histories, as suggested

33 by the results of hierarchical clustering of eye lens isotope values, due to the short distance between the two locations.

The SPO is much further south than any previously hypothesized origin for the northward migrant D. gigas, and there are several challenges to such a hypothesis. The southern edge of the

SPO (6.8° S) is approximately 9000 km from the British Columbia collection location if following nearshore habitats. Assuming D. gigas could maintain a 37 km/d migration rate for the entire migration (Stewart et al. 2012), migration would take approximately 243 days, two-thirds of a putative one year lifespan. Such a long migration would also not allow for an large amount of variability in migratory history, as seen in the squid collected from the coastal US, also making a long migration from the SPO seem unlikely. Instead patterns seen in squid eye lenses would be expected to mirror the pattern of isotopic variation with latitude, which is not observed here. Ad- ditionally D. gigas populations north and south of 5-6° N show mild genetic divergence, making it seem unlikely that these squid would regularly migrate northward from south of the equator

(Staaf et al. 2010).

There are a number of factors that could potentially be seen to confound our results. It is possible that there are undetected differences between muscle and eye lens δ13C values due to the low number of replicates examined (n=7) or that the results of no difference between muscle and eye lens δ13C values of Oregon squid is not generalizable to squid from other locations. These seem unlikely to unduly influence the results presented here. Systematic differences between muscle and eye lens were readily detected in δ15N values between muscle and eye lens, so any possible undetected difference in δ13C values would likely be small. Also, it appears that northern migrant squid are well mixed in terms of migration histories (this study), and therefore, is un-

34 likely to be significant differences between squid collected in California, Oregon, and British Co- lumbia. It is also conceivable that the low δ13C values of natal eye lens layers in this study is due to a far offshore origin of squid (> 1000 km), as squid collected offshore have been demonstrated to have lower δ13C values than those collected nearshore (Ruiz-Cooley and Gerrodette 2012;

Argüelles et al. 2012). This also seems unlikely as the drop in δ15N values of squid collected offshore is even more pronounced, while the δ15N values of natal eye lens layers in this study is relatively high (Argüelles et al. 2012; Ruiz-Cooley and Gerrodette 2012).

In this study we have presented data that indicates that the northward migrant Dosidicus gigas collected from various locations have variable migration histories, that are not necessarily more similar to squid collected near them than to squid collected at other locations. This suggests that D. gigas shoals are not stable entities over the course of the lifetime of the squid, which agrees with previous research over shorter time periods. Additionally the exceptionally low δ13C values found in natal eye lens layers of northern migrant D. gigas suggests that these squid originated from one of two bands of latitude: 1.6°-6.8° S or 37.6°-46.1° N. The northern possible origin appears to be the more likely because of shorter migration distances and better likelihood to explain observed variability in migration histories.

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42 Figure 1: A: Relationship between the diameter of the eye lenses of seven Humboldt squid (Dosidicus gigas) and their wet mass as successive outer layers are removed (grey open circles) and six whole dried eye lenses (black closed circles). B: Relationship of the dry mass composition of the eye lens of a Humboldt squid and the position within the lens (0% is the center of the lens, 100% is the outermost margin). The relationship is described by the natural log function shown in graph.

43 Figure 2: Hierarchical clustering dendrograms (average-linkage agglomerative, euclidean distance) of outer 8.4 mm of eye lens stable isotope values of Dosidicus gigas. Tips are shaded according to collection location. LOESS regression track of eye lens stable isotope values are shown below tips with values included in the analysis shown in black, those excluded shown in grey.

44 Figure 3: Comparison of isotope value of natal Dosidicus gigas eye lens layers to published values. Vertical gray lines are ranges of published values of muscle isotopic values by latitude, while the black solid line is the LOESS regression of those values. Horizontal broken lines are mean isotopic values of natal eye lens (2 mm) for squid in this study grouped by collection location. Nitrogen isotopic values corrected to equivalent muscle values (+2.03 ‰). Histograms represent distribution of all stable isotope values for all lens layers for all squid collected in northern latitudes in this study (squid collected in the Sea of Cortez are excluded)

45 Supplementary Figure 1: Example of sequence of nitrogen stable isotope values in right (black) and left (white) eye lens layers of Dosidicus gigas. Line is LOESS smoothed trendline (span=0.3)

46 Supplementary Figure 2: Eye lens layer carbon stable isotope patterns (LOESS smoothed) for all Dosidicus gigas used in this study.

47 Supplementary Figure 3: Eye lens layer nitrogen stable isotope patterns (LOESS smoothed) for all Dosidicus gigas used in this study.

48 Supplementary Figure 4: Carbon and nitrogen stable isotope values for sequential eye lens layers of individual Dosidicus gigas (Grey circles) compared to most common prey items (Black circles, mean +- SE). Grey circles are proportional in diameter to the diameter of the eye lens at each layer. Dosidicus gigas isotope composition values have been corrected for trophic enrichment (-3.3‰ δ15N) and for difference from muscle (+2.03‰ δ15N). Values for Euphasia pacifica (EUPA), Thysanoessa spinifera (THSP), Clupea pallasi (CLPA), Merluccius productus (MEPR), Engraulis mordax (ENMO), Diaphus theta (DITH), Gonatus onyx (GOON), Stenobrachius leucopsarus (STLE), and Tarletonbeania crenularis (TACR) taken from literature.

49 Supplementary Figure 5: Carbon and nitrogen stable isotope values for sequential eye lens layers of individual Dosidicus gigas (Grey circles) compared to most common prey items (Black circles, mean +- SE). Grey circles are proportional in diameter to the diameter of the eye lens at each layer. Dosidicus gigas isotope composition values have been corrected for trophic enrichment (-3.3‰ δ15N) and for difference from muscle (+2.03‰ δ15N). Values for Euphasia pacifica (EUPA), Thysanoessa spinifera (THSP), Clupea pallasi (CLPA), Merluccius productus (MEPR), Engraulis mordax (ENMO), Diaphus theta (DITH), Gonatus onyx (GOON), Stenobrachius leucopsarus (STLE), and Tarletonbeania crenularis (TACR) taken from literature.

50 Supplementary Figure 6: Carbon and nitrogen stable isotope values for sequential eye lens layers of individual Dosidicus gigas (Grey circles) compared to most common prey items (Black circles, mean +- SE). Grey circles are proportional in diameter to the diameter of the eye lens at each layer. Dosidicus gigas isotope composition values have been corrected for trophic enrichment (-3.3‰ δ15N) and for difference from muscle (+2.03‰ δ15N). Values for Euphasia pacifica (EUPA), Thysanoessa spinifera (THSP), Clupea pallasi (CLPA), Merluccius productus (MEPR), Engraulis mordax (ENMO), Diaphus theta (DITH), Gonatus onyx (GOON), Stenobrachius leucopsarus (STLE), and Tarletonbeania crenularis (TACR) taken from literature.

51 CHAPTER THREE

A SPONGE-SCALLOP MUTUALISM MAY BE MAINTAINED BY

OCTOPUS PREDATION

KIRT L. ONTHANK1†, BRIANNA G. PAYNE2, ASHLEY R. GROENEWEG2, THOMAS J. EWING3, NICOLAS MARSH3, DAVID L. COWLES3 and RAYMOND W. LEE1

1Washington State University School of Biological Sciences PO Box 644236, Pullman, WA 991644236

2Andrews University Department of Biology Price Hall 216, Berrien Springs, MI 491040410

3Walla Walla University Department of Biological Sciences 204 S. College Avenue, College Place, WA 99324

Formated for: Journal of Molluscan Studies

†Correspondence: K.L. Onthank email: [email protected]

52 ABSTRACT In the northeast Pacific ocean, valves of the scallop Chlamys hastata are often encrusted with sponges. This putative mutualism has generally been thought to protect the scallops from sea star predation, but supporting empirical evidence is weak. Scallops escape from sea stars by swimming and may rarely fall prey to them in nature. Consequently, a clear benefit to the scallop is lacking. We propose that octopus predation could provide the selective pressure to maintain this relationship. This hypothesis predicts that: (1) octopuses are an important cause of scallop mortality and (2) octopuses are less likely to consume scallops with sponges than those without.

We found that Chlamys hastata may comprise as much as one-third of the diet of the giant

Pacific octopus (Enteroctopus dofleini) and that E. dofleini is more than twice as likely to choose an unencrusted scallop as an encrusted one. While scallops are a smaller portion of the diet of the

Pacific red octopus (Octopus rubescens), this species is five times as likely to consume scallops without sponge encrustment as those with it. These findings provide strong evidence that octopuses provide an important selective pressure that maintains the scallop-sponge association.

Keywords: Enteroctopus dofleini, Octopus rubescens, Chlamys hastata, Myxilla incrustans, mutualism, predation.

53 INTRODUCTION The evolution and maintenance of interspecies interactions is one of the central questions of ecology. Long-term, close interspecific interactions, known as symbioses, fall into three categories: mutualisms, in which both participants benefit from the interaction; parasitisms, in which one participant benefits and the other is harmed; and commensalisms, in which one participant is benefited and the other is neither benefited nor harmed (Paracer & Ahmadhian,

2000). Of special interest is the maintenance and stability of mutualisms and how these interactions do not become exploitive (Bronstein, 2009). In particular, associations between bivalves and their epiobionts have been shown to shift between mutualistic and parasitic depending on the interactions between epibionts and predators on the bivalves (Dougherty &

Russell, 2005). Additionally, mutualisms that are based primarily on an exchange of services appear to be far less common than those that involve an exchange of resources (Ollerton, 2006), making the evolution and maintenance of bivalve-epibiont mutualisms of particular interest.

One such putative mutualism is that between scallops and sponges that frequently occur growing upon their valves, primarily their left valve which faces upward in resting scallops

(Bloom, 1975). Epibiotic sponges appear to benefit due to decreased predation by nudibranchs and by decreased mortality due to sediment accumulation (Bloom, 1975; Burns & Bingham,

2002).The scallops are thought to benefit principally through decreased predation by sea stars

(Bloom, 1975; Farren & Donovan, 2007). Sponge covering on scallops may reduce predation by providing visual or tactile camouflage, increasing handling costs to the predator by making grasping the scallop more difficult, or irritating to the predator with toxins or the spicules produced by the sponge (Laudien & Wahl, 2004).

54 Scallops appear to be structurally and behaviourally adapted to encourage sponge growth on their valve as increased sculpturing of scallop valves appears to be associated with increased sponge encrustment (Beu, 1965). Though they are most common epibiont on scallop valves, sponges are absent from the valves of other epifaunal bivalves in the same area such as Modiolus mussels (Lescinsky, 1993). Scallops swim when disturbed by sponge predators that pose no threat to the scallop (Bloom, 1975). Additionally, scallops appear to incur no increased energetic cost in swimming when encrusted with sponge, but do when covered by other epibionts

(Donovan et al., 2002). Costs incurred by the sponge have not been examined but may include exchanging a larger substrate for a safer, mobile substrate, or producing secondary metabolites irritating to scallop predators. The sponge-scallop association is known to occur from diverse locations such as the United Kingdom (Forester, 1979), New Zealand (Beu, 1965), Australia

(Chernoff, 1987), and the western coast of North America (Bloom, 1975). Despite the ubiquity of this association only cursory effort has been made to identify the selective pressures that maintain it.

In the Salish Sea, which includes the marine waters of the Strait of Georgia, Juan de Fuca

Strait, Puget Sound and all connecting waterways, the scallops Chlamys hastata (Sowerby, 1842) and Chlamys rubida (Hinds, 1845) often have valves covered by one of two species of sponge,

Mycale adhaerens (Lambe, 1894) or Myxilla incrustans (Esper, 1805). Scallops in this area are commonly found either with or without valve-encrusting sponges (pers obs). Protection from seastar predation, however, does not appear to be a significant selective pressure making it adaptive for Salish Sea scallops to maintain the symbiosis with the sponges. Even when scallops are abundant seastars in the wild rarely predate on scallops with or without encrusting sponges

55 (Table 1)(Mauzey et al., 1968; Shivji et al., 1983). In laboratory tests sponge encrustment has strongly and consistently deterred only Evasterias troschelii from engaging in scallop predation

(Table 1) (Bloom, 1975; Farren & Donovan, 2007). The lack of evidence that sea stars consume an appreciable number of C. hastata or C. rubida in the wild, along with the finding that only

Evasterias troschelii is readily deterred by sponge encrustment makes it appear unlikely that sea star predation could alone provide the selective pressure to maintain this association. Some other ecological pressure may be responsible for the maintenance of this relationship.

Octopuses have predated on scallops since at least the Pliocene, (Bromley, 1993; Harper,

2002) and we have found middens, piles of discarded prey parts found at the entrances of octopus dens, of the giant Pacific octopus, Enteroctopus dofleini (Wülker, 1910) in the Salish Sea containing abundant C. hastata and C. rubida valves (pers obs). Previous investigators recognised that E. dofleini consumes C. hastata and C. rubida, but considered the more abundant sea stars to have a greater impact (Bloom, 1975). However, the impact of octopuses as predators was likely underestimated due to their cryptic nature. Additionally, the metabolic rate of benthic octopuses far exceeds that of seastars (Seibel, B.A. & Drazen, J.C., 2007); if scallops compose even a modest portion of octopuses' diets, their total consumption may surpass that by seastars.

The east Pacific red octopus, Octopus rubescens (Berry, 1953), abounds within the range of C. hastata's range and also consumes C. hastata. Although scallops do not appear to be featured as prominently in the diet of O. rubescens as in the diet of E. dofleini (Anderson et al.,

1999), even moderate consumption by the abundant O. rubescens could exert important selective pressures on the scallop-sponge symbiosis.

In the Salish sea Chlamys scallops occur in discrete aggregations, or beds, with scallop

56 densities of these beds reaching 11.8 scallops/m2 (Lauzier & Bourne, 2006; Surry et al., 2012)

Individuals occur with widely varying amounts of encrusting sponge, or without sponge (pers obs). If a predator encountering a bed of scallops preferentially consumes those unencrusted or lightly encrusted over those fully encrusted, that predation pressure would likely help maintain this sponge-scallop association. Additionally, even if the presence of the sponge does not change the total number of scallops that the predator consumes, if it shifts predation toward unencrusted scallops the sponge would benefit their host scallops by decreasing the probability of that individual being consumed.

Here we test the hypothesis that octopus predation plays a role in maintaining the association between the sponge M. incrustans and the scallop C. hastata, the most common sponge-scallop association in the Salish Sea. Two predictions must be supported for this hypothesis to be tenable: 1) Octopus predation must be an important cause of scallop mortality in the wild and 2) octopuses must show a preference for scallops with less or no sponge on their valves. While it would be difficult to determine directly if octopus predation is an important source of scallop mortality in situ, we set forth here to test the prediction that scallops are an important octopus prey item in the Salish Sea, thereby implicating octopuses in this role.

Additionally we hypothesised that sponges interfere with an octopus' ability or likelihood to grasp and open the scallop. There are at least three possible mechanisms by which sponge encrustation on scallops may deter predation by octopuses: 1)Visual and/or tactile camouflage, 2) interference with grasping and handling by octopuses, or 3) irritation by sponge spicules or secondary metabolites of sensitive octopus tissues such as suckers (Wells, 1963; Wells, Freeman

& Ashburner, 1965), or the mouth parts. Octopuses regularly follow a stereotyped progression of

57 strategies to gain entry to bivalve prey, first attempting to pull the valves open, then drilling the shell to inject venom from the posterior salivary gland, and finally chipping at the margin of the valves with the beak (Anderson & Mather, 2007). Each additional strategy appears to require more time and effort by the octopus to open the bivalve. If sponges interfere with the octopus' ability to pull open a scallop, a higher rate of drilling should be observed in scallops consumed with sponges than those without.

MATERIALS AND METHODS

Determination of Diet Important components of the diet of the two species of octopus was estimated by two complimentary methods: midden analysis and eye lens stable isotope analysis. Middens of prey remains help determine the diet of octopuses and can provide easy identification of prey species present in the diet. However, this approach only reveals the recent diet of the octopus and suffers from biases due to differential rates of prey item removal and preservation in the middens

(Ambrose, 1983). Stable isotope analysis, especially of archival tissues such as cephalopod eye lenses, can provide a longer term record of diet and dietary shifts but generally cannot reveal individual prey species. Instead, stable isotopes distinguish larger groups of prey with different nutritional sources and trophic position (Hunsicker, M.E., Essington, T.E., Aydin, K.Y. & Ishida,

B., 2010). When used in conjunction these two methods provide a more complete picture of octopus diet than does either strategy alone.

Complete middens from eight E. dofleini dens ranging in depth from 6 to 15m were collected near Coffin Rocks, Fidalgo Island, Washington (48° 24' 46.7" N, 122° 39' 43.5" W)

(Fig. 4) by SCUBA during July and August 2005 and February 2006. Dens were initially located

58 by the midden and then confirmed to be a den by the presence of an octopus. The entire midden was collected and sorted into “old” and “new” shells according to (Dodge & Scheel, 1999) and

(Scheel et al., 2007). Shells identified as “old” by the presence of algal overgrowth on the interior of the shell were not counted. Crab carapaces recovered from the midden samples rarely survived transport from the den to the lab and were thus recorded while collecting the midden.

Prey items were identified using the key Marine invertebrates of the Pacific northwest (Kozloff,

1996) and the minimum number of individual prey in each midden was determined for each taxon by counting body parts and recording the highest number of unique body parts. For example if three right chelipeds, one left cheliped and four carapaces of Cancer productus were recovered in a single midden, four Cancer productus were recorded.

Twenty-one glass bottles with O. rubescens inside were collected from Admiralty Bay,

Whidbey Island, Washington (48° 9' 47.7" N, 122° 38' 12.0" W). Fifteen of these contained prey remains and were used for this study. After removal of the resident octopus all contents of the bottles were withdrawn and treated as were the items in the E. dofleini middens. Because the substrate at Admiralty Bay is dominated by fragments of barnacles, it was unclear whether barnacle fragments inside the glass bottles had merely fallen into the bottle. Barnacles were therefore not counted as part of the midden.

For stable isotope analysis, eye lenses of two E. dofleini were collected from individuals that were captured in north Puget Sound by fishermen and donated dead to the Seattle Aquarium, and a third E. dofleini was collected from Admiralty Beach. Stable isotope composition of the sequentially formed layers of cephalopod eye lenses can be used to reconstruct past changes in the diet of individual cephalopods (Hunsicker, M.E., Essington, T.E., Aydin, K.Y. & Ishida, B.,

59 2010). Lenses were stored frozen in a -20 °C freezer until sectioning was possible. Eye lenses were sectioned by first measuring the diameter of the eye lens with digital callipers and then peeling off of the lens layers in the outer approximately 0.5mm, hereafter referred to as a growth layer group (GLG). This process of removing successive GLGs was then repeated with the remainder of the lens until only the inner 0.2 mm remained, which was used as the final GLG.

Each GLG position in the lens was recorded as the diameter (mm) of the lens before the GLG was removed. For instance, in a lens with a total diameter of 15 mm, the first GLG removed would be 15 mm. Each successive layer removed would correspond to the smaller diameter of the remaining lens, approaching zero toward the centre. Excised GLGs were placed into separate microcentrifuge tubes and dried at 60 °C for at least 24 hrs.

For comparison of isotopic composition to that E. dofleini eye lenses, prey species of similar size to those found in middens, as well as the sponge M. incrustans, were collected at

Coffin Rocks and Cornet Bay, Whidbey Island, Washington (48° 23' 53.9" N, -122° 37' 49.4" W)

(Fig. 4). Consumable soft tissues of the prey items were removed and dried at 60 °C for at least

24 hours.

Dried eye lens and prey samples were ground to a fine powder and 0.50 mg was placed into a tin capsule for isotopic analysis of 13C/12C and 15N/14N ratios. Samples were analysed using a Eurovector elemental analyser in line with a Micromass Isoprime continuous flow stable isotope mass spectrometer. Results are presented in the standard δ notation, where isotopic ratios are expressed in ‰ differences relative to the conventional standard, the PeeDee Belemnite limestone for carbon and atmospheric nitrogen for nitrogen. Routine precision for δ13C and δ15N was +/- 0.1 ‰ and 0.3 ‰ respectively. For plotting purposes δ15N values of eye lens layers were

60 corrected for trophic enrichment by subtracting 3.3‰, based on trophic enrichment values measured in another cephalopod, Sepia officinalis (Hobson, K.A. & Cherel, Y., 2006), so that octopus isotopic values could be compared directly to those of prey items.

Preference Trials Four E. dofleini were collected at Coffin Rocks and Admiralty Beach, Washington by

SCUBA between June 2007 and June 2011. Octopuses were housed in 2840 l flow-through tanks

(3.0m2 footprint) with running seawater at 11°C and provided with appropriately sized dens.

Octopuses were acclimated to captivity for one week before trails began. Scallop preference trials occurred at night, which is thought to be the normal foraging time for this octopus (Mather,

Resler & Cosgrove, 1985). C. hastata scallops were collected by SCUBA near Northwest Island,

Washington (48° 25' 8.9” N, -122° 40' 16.1” W) (Fig. 4). Scallops that were visually estimated to have >80 % of the left valve covered with sponge were left with all epibionts on the valve

(encrusted scallops). Encrusted scallops were selected with minimal non-sponge epibionts

(visually estimated >5 %) and no epibionts that would likely be irritating to the octopus such as hydroids. Scallops visually estimated to have <80 % of the left valve covered with sponge were scrubbed with a wire brush to remove all sponges and other epibionts (clean scallops). For each trial 10 encrusted and 10 clean live scallops were piled in the centre of the octopus holding tank late in the evening. Scallops in each treatment were matched by size to scallops of the other treatment, and selected to approximately correspond to the size of scallop found in octopus middens. The next morning the number of encrusted and clean scallops that had been eaten was recorded. If any scallops had been eaten all were removed from the tanks until the next trial.

Octopuses were not fed between trials. To ensure that the octopus could feed to satiation on

61 whatever type of scallop was chosen, if the octopus consumed more than half the scallops in any given trial, at the next trial the total number of scallops presented was doubled, while still maintaining an equal number of encrusted and clean scallops.

During trials of three of the E. dofleini the position of drill holes on the valves of all consumed scallops were recorded. To standardise measurements of drill hole positions on valves of varying sizes the drill holes were measured on both the anterior-posterior and dorsal-ventral axes, scaled so that the anterior and dorsal margins were set to 0 and posterior and ventral margins were set to 100.

Six male O. rubescens ranging from 46 to 250 g were collected at 15 to 20 m depth at

Admiralty Bay between June and August 2007. These octopuses were transported to the Rosario

Beach Marine Laboratory and housed in 15 l flow-through tanks with running seawater at 11C.

Octopuses were allowed to acclimate to captivity for at least one week while being maintained them on a diet of Nuttallia obscurata (Mollusca:Bivalvia) and Hemigrapsus nudus

(Crustacea:Brachyura). After the acclimation period the preference of each O. rubescens between encrusted and clean scallops was tested. Trials were run during daylight hours from 0700 to

2100. In each trial, four encrusted and four clean live, size-matched scallops were placed in a 61 cm x 92 cm, 250 l rectangular tank. One encrusted and one unencrusted scallop was placed in the four corners of the tank immediately adjacent to each other so that the octopus would likely encounter both simultaneously. The octopus was placed into the centre of the tank and allowed to approach, select and consume a scallop. As soon as the octopus made a choice and consumed one scallop the octopus was removed, the scallops were replenished, and a trial was begun with a different octopus. Trials lasted until the octopus either chose a scallop, or had made no choice

62 within 2 h, at which time we changed octopuses. Each octopus was fasted for a period of at least

24 h before being used in another trial. Each octopus was tested until it made three choices.

Statistics Because the scallops were paired, the choices of one scallop type were not independent from choices for the alternate scallop type in this experimental setup. Therefore, in lieu of a student t-test, the Quade's test, a non-parametric analysis of ranks (Roa, 1992), was employed to compare octopus preferences. This test was performed using the base package in R (R

Development Core Team, 2008).

The L-function transformation of Ripley's K-function allowed us to compare the spatial distribution of drill holes on the scallop valves to a completely spatially random (CSR) Poisson distribution (Ripley, 1991). Ripley's K-function, K(r), is determined by the number of points within a distance (r) of each point multiplied by the inverse of the density of points. Therefore, the expected values of K(r) for a CSR distribution would be equal to the area within distance (r), or πr2. The L-function is equal to the expected value of K(r) less the observed value of K(r), yielding an expected value of L(r) to be 0 for any radius. Simplified, the L-function is calculated from K(r) as:

K r L r=r−  π Values of L(r) greater than zero indicate a regular (dispersed) distribution of points and those less than zero indicate a clustered distribution. Similarly an Lcross(r) function can be calculated for the distribution of points of two differing types using number of points of type 1 within distance (r) of each point of type 2, and the reverse. Values of Lcross(r) greater than zero indicate segregation of the point types, while values less than zero indicate aggregation. To determine if octopuses

63 drilled in a focused manner rather than randomly on the scallop valves, the pattern of actual drill hole locations on the valve was compared by 5000 bootstrap replicates of a CSR distribution to what would be expected if the drill holes were randomly distributed. Additionally, to determine if the presence of sponge on the valve changed the location of drill holes the Lcross(r) function was used to compare relative distributions of drill holes on the valves of encrusted and unencrusted scallops. The isotropic edge correction was used in both calculations. The R package Spatstat was used to calculate L(r), Lcross(r), and to generate CSR bootstrap replicates (Baddeley & Turner,

2005).

Meaningful groupings of prey-item stable isotope values were determined by model- based clustering using the MCLUST package in R (Fraley & Raftery, 2002). This analysis tool selects the optimal model and number of clusters using Bayes information criterion (BIC) and assigns individual points to these clusters.

RESULTS

Determination of natural diet C. hastata comprised over one third of the individual prey items identified in the middens of E. dofleini and was represented in all nine middens. In the middens of O. rubescens, the scallop C. hastata comprised only 9% by count but was still the fourth most common prey item recovered (Fig. 5, Supplementary Table 1).

Three clusters of prey isotopic values were identified, one containing Cancer productus

(Randall, 1839) and Pugettia gracilis (Dana, 1851) and hereafter referred to as the “crab cluster”, one containing Chlamys hastata, Chlamys rubida (Hinds, 1845), Calyptraea fastigiata (Gould,

1846), Balanus nubilis (Darwin, 1854), Clinocadium nuttallii (Conrad, 1857), Saxidomus

64 gigantea (Deshayes, 1839), Humilaria kennerlyi (Reeve, 1863), and Terebratalia transversa

(Sowerby, 1846) and hereafter referred to as the “filter feeder cluster”, and one containing

Myxilla incrustans and Acmaea mitra (Eschscholtz, 1833), hereafter referred to as the

“sponge/acmaea cluster” (Fig. 6). Uncorrected E. dofleini δ15N values ranged from 10.7‰ to

14.8‰ and δ13C values ranged from -18.4‰ to -15.4‰.

Preference trials In preference trials the four E. dofleini chose clean scallops significantly more often than sponge-encrusted scallops (Quade test, Quade F = 15, numerator df = 1, denominator df = 3, P =

0.03047) as did the six O. rubescens (Quade test, Quade F = 30.625, numerator df = 1, denominator df = 5, P = 0.002643) (Table 2). Individual octopuses varied in preference, especially E. dofleini, which ranged from a preference of unencrusted:encrusted of 1.2:1 to 3.3:1, with an average of 2.1:1. For the three E. dofleini with the presence and distribution of drill holes recorded, drill holes were found significantly more often on sponge-encrusted scallops than predicted if the octopus were equally likely to drill clean and encrusted scallops (χ2 with Yates's correction= 11.572, df = 2, P = 0.003) (Table 2). Ripley's L-function of the spatial distribution of the drill holes indicated the drill holes were not randomly distributed, but clustered on the valve

(Fig. 4B). This clustering of drill holes was near the adductor muscle. Additionally the Lcross- function suggests drill holes from sponge-encrusted valves and clean valves were aggregated in the same area of the valve, indicating that the spatial distribution of drill holes did not change between valve types (Fig. 4C). Octopuses did not significantly change the distribution of drill holes between left and right valves due to presence of sponge (Clean 11 right:1 left, Encrusted 18 right:5 left, χ2 with Yates's correction = 3.80, df=1, P=0.0513).

65 DISCUSSION Middens collected from the dens of E. dofleini and O. rubescens indicated that the scallop, C. hastata comprises a substantial portion of the normal diet of these octopuses in the

Salish Sea (Fig. 5). This is in contrast to seastars, which consume very few scallops (Mauzey et al., 1968). Previous studies at a variety of locations have produced mixed results regarding the importance of Chlamys spp. to the diet of this octopus. Investigations in Alaska found scallops to be of minimal dietary importance in intertidal regions where most octopuses were found, but scallops composed 60 % of midden items at depths greater than 10m (Vincent, Scheel & Hough,

1998). A second investigation of E. dofleini middens in Alaska found that Chlamys spp. comprised 6.7 % of all prey items in intertidal middens despite Chlamys spp. not being located in live prey intertidal quadrats, suggesting that E. dofleini may seek out this genus of scallops even when locally scarce (Scheel et al., 2007). Two investigations in British Columbia found that C. hastata composed only 1 % of the total midden (Cosgrove, 1989; Hartwick, Tulloch &

MacDonald, 1981). Even if C. hastata composes a modest portion of the diet of these octopuses, the much greater metabolic rate of octopuses than sea stars (Seibel, B.A. & Drazen, J.C., 2007) and consequently higher consumption rate suggest that an octopus may consume many more scallops than would a comparably sized sea star. Additionally, octopuses may be more abundant in benthic habitats than is commonly realised. During this study the authors encountered as many as 16 different O. rubescens on a single one-hour dive in Admiralty Bay, and at Coffin Rocks could regularly locate up to four large (visually estimated >10 kg) E. dofleini within a 50 m2 area. A population survey of E. dofleini in Alaska found an average of one octopus per 3,700 m2 in intertidal areas, with one site yielding one octopus per 323 m2 in 1996 (Scheel et al., 2007).

Stable isotope composition results suggest two E. dofleini preyed heavily on filter feeders

66 early in life but later shifted to a more crab-based diet (Fig. 6). A third younger E. dofleini had a stable isotope composition that was most closely associated with the filter feeder group throughout its lifetime. These data are consistent with the prediction that filter feeders are an important component of E. dofleini diet, especially at small octopus sizes. Of the filter feeder prey in middens of E. dofleini, C. hastata was the most numerous (Fig. 5). Proportions of prey items in middens can be biased by differential removal or destruction of prey parts. Because C. hastata has one of the lightest shells of filter feeders, they would likely be removed from the middens first and therefore be under-represented. Octopuses are likely important predators on scallops in the Salish Sea, and may in fact be more important scallop predators than are sea stars.

In preference trials both octopus species showed a significant preference for clean scallops, this preference being more pronounced in O. rubescens than in E. dofleini (Table 2).

Conceivably, removing sponges from the valves may have biased octopus preference for reasons other than the absence of sponge. Destructive removal of sponges may have liberated irritating secondary metabolites which may have remained on the valves or the increased handling time of these scallops may have caused the scallops to remain closed for a longer time once introduced to the octopus' aquarium. Both of these potential biases, however, would likely bias octopus preference against artificially cleaned scallops, and therefore not likely to change the results of the preference trials.

In preference trials E. dofleini were significantly more likely to drill encrusted scallops than clean scallops, indicating a higher rate of failed pulling attempts. This supports the hypothesis that the encrusting sponge interferes with attempts by the the octopus to pull the scallop open. Because drilling scallops requires longer handling times, this simultaneously does

67 not support the hypothesis that the sponge causes irritation to the octopus. Additionally, while drill holes were focused on the valves, the location of drill holes did not significantly differ between clean and encrusted scallops, nor did octopuses avoid drilling the more heavily encrusted left valves (Fig. 4), indicating octopuses were not attempting avoid irritation.

It is possible that increased clean scallop mortality in our experimental setting does not reflect increased mortality in situ, but we think this is unlikely. Octopuses will encounter scallop beds with similar densities of scallops as our experimental settings (6.8 scallops/m2 in E. dofleini trials, 11.8 scallops/m2 in O. rubescens trials, up to 11.8 scallops/m2 in situ (Surry et al., 2012)), and with varying levels of sponge encrustation, strongly suggesting that choices by octopuses in our trials reflect differential rates of predation in the wild.

Similar sponge-scallop associations have been identified, such as that between Chlamys varia and Halichondria panicea in England (Forester, 1979), between Chlamys opercularis and two sponges in the genus Suberites in the Scottish Sea (Pond, 1992), and between Chlamys asperrima and crellid and myxillid sponges in Australia (Chernoff, 1987) (Table 3). Each of these associations was characterised as benefiting the scallop by preventing sea star predation, though in only one of these instances were sea stars shown to consume a substantial number of scallops. In that instance C. asperrima composed approximately 10% of the diet of the sea star

Coscinasteria calamaria and sponge encrustation was shown to confer some protection (Pitcher

& Butler, 1987). The primary protection for C. asperrima, however, may be its habitat choice, since it often attaches itself by byssal threads to hard substrates above the sea floor where

Coscinasteria calamaria seldom forages (Chernoff, 1987). Additionally, Chlamys bifrons, a scallop occurring in the same geographic area as C. asperrima, does not harbour encrusting

68 sponges, yet in laboratory conditions it has a low rate of predation by Coscinasteria calamaria similar to that of sponge-encrusted C. asperrima (Pitcher & Butler, 1987). C. bifrons evades sea star predators by being much more easily provoked into a swimming escape response. Previous work has also shown that Octopus vulgaris predation on the sympatric arcid bivalve Arcoa noae in the lab decreased when the scallop was coated with the sponge Crambe crambe. However, this study failed to demonstrate whether O. vulgaris preyed on A. noae in the wild (Marin & López

Belluga, 2005). For none of these sponge-scallop associations has the possibility of octopus predation as a mechanism to maintain the association been rigorously explored.

Conclusion

The potential role of octopus predation in maintaining the putative mutualism between

Chlamys scallops and sponges has remained unevaluated until now, with attention paid solely to the potential role of seastar predation. In the case of C. hastata and C. rubida in the Salish Sea, asteroids do not appear to be an important predator. The data presented in this study are consistent with the predictions that O. rubescens and E. dofleini include a substantial number of

C. hastata in their diet, and that these octopuses preferentially consume scallops unencrusted by sponges. Although a preference for unencrusted scallops in a lab setting does not necessarily mean a higher mortality rate for these scallops in situ due to octopus predation, the abundance of scallops in discrete beds and variable level of sponge encrustation within these beds strongly suggests that octopuses in the field are presented with choices comparable to our lab settings, and will likely make similar choices in situ. These findings suggest octopus predation as an important selective factor, possibly even more significant than sea star predation, in maintaining the C. hastata – M. incrustans association, although other factors may also play a role. Future studies

69 should endeavour to determine how octopus predation influences scallop populations in situ and expand this work both geographically and to other sponge-scallop associations.

ACKOWLEDGEMENTS I thank JG Galusha and JR Nestler for their valuable input on this manuscript. I thank EA Verde and John Mayberry for help collecting and processing octopuses and prey items. I thank RC

Anderson and the Seattle Aquarium for providing two frozen Enteroctopus dofleini for this research. I also thank the two anonymous reviewers and Dr. Janet Voight for valuable advice in revising this manuscript. This project was supported by funds from the Walla Walla University

Department of Biology and the Phillip and Neva Ableson Fellowship and Carl Elling Award from Washington State University.

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76 Table 1. Summary of literature records of a significant reduction in seastar predation on scallops with sponge encrusted valves over scallops without sponges (“yes” means reduction was demonstrated, no means it was not). Additionally, the portion of diet consisting of of scallops for each seastar species obtained from in situ observations is included (Mauzey et al., 1968).

Bloom 1975 Farren & Donovan 2007 % diet Seastar species Myxilla incrustans Mycale adherens Pycnopodia helianthoides no no yes 0 Evasterias troschelii yes – – 0.22 Pisaster ochraceus no – – 0 Orthasterias koehleri no – – 1.27 Pteraster tesselatus – – – ? (2 obs) Crossaster papposus – – – 0.75

77 Table 2. Number of clean and sponge-encrusted scallops consumed by four Enteroctopus dofleini and six Octopus rubescens during prey choice trials. Choices were significantly different for each prey type in both E. dofleini (Quade test, Quade F = 15, num df = 1, denom df = 3, P = 0.03047) and O. rubescens (Quade test, Quade F = 30.625, num df = 1, denom df = 5, P = 0.002643). Drill holes on scallops consumed by E. dofleini differed significantly from a random distribution (χ2 with Yates's correction= 11.572, df = 2, P = 0.003), with a greater than expected number of drill holes in the encrusted scallops.

Consumed Drilled Mass Octopus (kg) Clean Sponge Clean Sponge Enteroctopus doleini 07 20.55 34 14 – – E. dofleini 09 8.40 33 10 8 7 E. dofleini 11m 0.66 22 14 1 4 E. dofleini 11f 0.30 16 13 3 12 Octopus rubescens 1 0.10 3 0 – – O. rubescens 2 0.15 3 0 – – O. rubescens 3 0.05 2 1 – – O. rubescens 4 0.18 3 0 – – O. rubescens 5 0.25 2 1 – – O. rubescens 6 0.16 3 0 – –

78 Table 3. Summary of known associations between Chlamys genus scallops and sponges.

Known Symbionts Putative Predators Scallop Sponge Location Scallop Sponge Reference New Chlamys dieffenbachi ? ? ? Beu 1965 Zealand Archidoris Chlamys hastata & Myxilla incrustans & Northeast montereyensis See Table 1 Bloom 1975 Chlamys rubida Mycale adherens Pacific & Anisodoris nobilis Halichondria Asterias rubens panicea, Mycale Chlamys varia England & Marthasterias ? Forester 1979 macilenta & glacialis Hymedesmia pansa Chernoff 1987, Crellids and South Coscinasterias Chlamys asperrima ? Pitcher & Myxillids Australia calamaria Butler 1987 Archidoris Chlamys opercularis Suberites sp. Scottish Sea Asterias rubens Pond 1992 pseudoargus

79 Figure 4: Locations of collection sites for Enteroctopus dofleini, Octopus rubescens and octopus prey items. Salish Sea has been denoted by light grey in insert, all other marine waters in dark grey.

80 Figure 5: Treemap of midden contents of 15 Octopus rubescens middens and 8 Enteroctopus dofleini middens. Relative number of each prey species found in all middens is represented by the area of the box, while the shading of the box represents the proportion of middens in which prey species was observed. Each prey species is then situated hierarchically into higher taxonomic groups so that the relative representation of these higher groups in the middens can be easily compared. Prey species composing less than 5% of midden items are lumped into Other categories within each high taxonomic category. Plot was generated using the tmPlot function from the ‘treemap’ package for R (R Development Core Team, 2008). For prey item counts for all prey species recovered from each den see Table S1.

81 Figure 6: LOESS smoothed nitrogen and carbon stable isotope composition series of the eye lens layers of three Enteroctopus dofleini (right) collected in the Salish Sea. E. dofleini δ15N values were corrected for trophic enrichment by subtracting 3.3‰. Diameter of bubble corresponds to the diameter at which eye lens layer was collected, therefore larger bubbles reflect isotope composition from later in life. Common prey items are also plotted by initials (Cp=Cancer productus, Pg=Pugettia gracilis, Bn=Balanus nubilus, Ch=Chlamys hastata, Cr=Chlamys rubida, Hk=Humilaria kennerlyi, Sg=Saxidomus gigantea, Cn=Clinocardium nuttallii, Cf=Calytrea fastigiata, Am=Acmaea mitra, Tt=Terebratalia transversa, Mi=Myxilla incrustans). Three potential prey-item clusters determined by model- based cluster analysis are identified by boxes.

82 Supplemental Table 1: Counts of prey (minimum number of individuals) recovered from eight Enteroctopus dofleini middens (ED1-8) and fifteen Octopus rubescens middens (OR1-15) 8 3 Species ED1 ED2 ED3 ED4 ED5 ED6 ED7 ED8 ED TOT OR1 OR2 OR3 OR4 OR5 OR6 OR7 OR8 OR9 OR10 OR11 OR12 OR13 OR14 OR15 OR TOT Chlamys hastata 15 45 19 20 27 21 7 4 158 0 2 0 2 0 2 5 1 0 1 0 0 0 0 1 14 Cancer productus 4 13 7 13 8 2 0 1 48 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Glebocancinus oregonensis 18 1 3 1 1 0 0 1 25 2 1 0 0 0 1 1 0 0 0 0 0 1 0 1 7 Pugettia gracilis 4 2 5 2 2 4 0 0 19 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Metacarcinus gracilis 0 0 0 3 1 2 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Metacarcinus magister 0 0 1 0 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Hapalogaster mertensii 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 unid crab (pincer) 2 1 1 0 2 1 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Terebratalia transversa 4 14 6 1 3 2 0 0 30 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Laqueus californianus 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acmaea mitra 4 2 4 1 2 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Calyptraea fastigiata 1 2 2 2 4 1 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Crepidula spp. 3 2 4 4 1 5 0 0 19 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 3 Diodora aspera 1 1 0 0 1 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Mytilus sp. 0 3 0 1 0 2 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Saxidomus gigantea 6 5 9 15 3 9 0 0 47 0 1 1 0 0 3 3 0 0 1 0 0 3 0 0 12 Clinocardium nuttallii 0 1 0 11 1 7 0 1 21 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 2 Macoma nasuta 1 1 2 1 0 2 0 0 7 0 0 2 0 0 0 0 0 0 0 0 0 0 0 1 3

8 Panopea abrupta 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 Strongylocentrotus droebachiensis 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 Strongylocentrotus franciscanus 0 0 1 1 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pododesmus macroschisma 1 3 0 17 1 2 0 0 24 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Katharina tunicata 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Cryptochiton stelleri 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Tonicella lineata 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Petrolisthes eriomerus 0 0 0 0 0 0 0 0 0 0 1 0 0 0 8 9 2 0 2 1 0 0 0 3 26 Nucella lamellosa 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28 5 0 1 0 0 3 2 1 1 41 Oregonia gracilis 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 Lophopanopeus bellus 0 0 0 0 0 0 0 0 0 0 0 4 0 1 10 4 0 0 1 1 1 2 0 2 26 Humilaria kennerlyi 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 2 Cranopsis cucullata 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 Calliostoma ligatum 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 2 Leukoma staminea 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 1 4 Mya arenaria 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 2 Euspira sp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 Colus griseus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Scyra acutifrons 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 65 97 66 95 59 60 7 8 457 4 7 8 2 1 53 28 3 1 9 2 5 14 4 11 152 CHAPTER FOUR

RARE CAPTURE OF HYDROTHERMAL VENT-ASSOCIATED OCTOPUS

REVEALS SURPRISING INFORMATION ABOUT DIETARY HISTORY

Kirt L. Onthank* and Raymond W. Lee

School of Biological Sciences, Washington State University

PO Box 644236, Pullman, WA 99164-4236

*-corresponding author

Email: [email protected]

Formatted for: Marine Biology

85 ABSTRACT Deep-sea hydrothermal vent habitats support chemosynthetically based ecosystems that are, as of yet, poorly understood. As with many shallow marine habitats, hydrothermal vent ecosystems appear to be partially structured by predation. However, specific predators are not readily known, and may include non-vent-endemic fauna. Graneledone genus octopuses are known to associate with vents, but are not endemic to those habitats. Extraordinarily little is known about the diet of the Graneledone that aggregate near vent systems, but evidence from the gut contents of a single individual has indicated these octopuses rely heavily on vent-endemic fauna, potentially filling the role of major vent predator. The rare collection of a vent-associated

Graneledone cf. boreopacifica has enabled stable isotope analysis of an archival tissue, eye lens, to determine vent fauna contribution to the diet over the course of the lifetime of the octopus, considerably increasing our knowledge about diet of this species. Results indicate that vent fauna contributed little, if any, biomass to the Graneledone diet at any life stage, despite having access to nearby productive vent habitats. Instead of providing and abundant food supply vents may supply solid substrate for egg-brooding, with octopuses only consuming sulfide-laden vent fauna when non-vent fauna is not readily available.

86 INTRODUCTION Deep-sea hydrothermal vents are a recently discovered, globally distributed habitat that supports high biomass ecosystems deriving primary production from chemosynthesis rather than photosynthesis (Jannasch & Wirsen 1979; Lonsdale 1977). Largely due to extreme difficulty in accessing these habitats and their recent discovery, the food webs and trophic interactions of the biota supported by the vents had not been extensively characterized, and in particular how vent and abyssal food webs may be connected (Bergquist et al. 2007). Stable isotope analysis has proven to be a valuable tool in characterizing trophic interactions in these habitats, as deep-sea photosynthetically based food webs are largely distinct in carbon and nitrogen isotopic composition from deep-sea chemosynthetically based food webs (Van Dover 2007).

Despite this gap of knowledge, exclusion experiments have demonstrated predation significantly impacts the structure of hydrothermal vent communities, primarily by removing small grazers such as gastropods and allowing settlement and recruitment of the sessile invertebrates, such as vestimentiferan tube worms, that provide the basic physical structure of many vent communities (Micheli et al. 2002).

One potential predator on hydrothermal vent fauna in the North Pacific are octopuses in the genus Graneledone. Graneledone cf boreopacifica* are commonly found near, though not limited to, hydrothermal vents in the Juan de Fuca Ridge vent system, and preliminary evidence strongly suggests that octopuses in this genus have at least minimal tolerance for the high sulfide

* Two species in the genus have Graneledone have been described from the north Pacific: G. boreopacifica (Nesis, 1982), with the type specimen from the Sea of Okhotsk at a depth of 1350m and G. pacifica (Voss & Pearcy, 1990), which the type specimen being collected from the northeast Pacific at a depth of 2706 m. Species were later synonymized with the name G. boreopacifica taking precedence (Hochberg 1998), However, several characters still suggest two species may be present (Voight & Grehan 2000). If there are two species existing in the northeast Pacific, the species collected here is G. pacifica; if one species exits then the name would be G. boreopacifica. For the duration of this manuscript I will refer to the octopus collected here at Graneledone cf. boreopacifica

87 and low oxygen present in these environments (Voight 2000b). Although not endemic to chemosynthetic ecosystems, this species will aggregate in large numbers near these habitats in the north Pacific leading several investigators to hypothesize that octopuses are attracted to these sites due to high biomass of potential prey items (Juniper et al. 1992; Mottl et al. 1998).

Exceedingly little is known about the dietary habits of this potentially important hydrothermal vent predator (Voight 2008). Collection of octopuses is difficult at any depth, however the difficulty of accessing hydrothermal vent habitats compounds this difficulty. Additionally, guts of octopuses in this genus often contain little (Voight 2008) and gut contents have been obtained from only four individuals associated with hydrothermal vents with the gut of one individual found with a large number of vent-endemic gastropods and polychaetes (Voight 2000a; Voight

2012). Graneledone have larger beaks than related taxa, presumably for crushing shells of prey, enabling this genus to consume vent-endemic grazers (Voight 2000a). Combine with information that octopuses often have higher mass-specific metabolic rates than most other benthic predators, including fish (Seibel & Drazen 2007), relatively larger size than other potential predators on vent-endemic grazers (Desbruyères et al. 2006) indicates that Graneledone predation may be an important component in structuring hydrothermal vent biotic communities. Limited dietary observations, however, cripple our ability to assess the level of Graneledone participation in hydrothermal vent food webs.

Stable isotope analysis of tissues enables ecologists to trace nutritional contributions from sources distinct in stable isotope composition, such as hydrothermal vent and abyssal habitats

(Van Dover 2007). Specifically nitrogen stable isotopes are distinct between vent and abyssal food webs with vent fauna ultimately deriving N2 from mid-ocean ridge basalts (via superheated

88 vent fluids), which have been documents to have very low δ15N values (-3.3 ± 1.0 ‰, Marty &

Zimmermann 1999), and vent fauna being correspondingly 14N rich (Bergquist et al. 2007;

Levesque et al. 2003; Limén et al. 2007; Van Dover & Fry 1994). Abyssal fauna, by contrast,

15 derive the bulk of their nitrogen from atmospheric N2, which averages 0 ‰ δ N by convention, and oceanic nitrate, which averages 4-5 ‰ δ15N (Sigman et al. 1997), incorporated into organic material near the ocean surface by photosynthetic primary producers. This organic nitrogen then sinks towards the ocean floor, during which it is repeatedly subjected to consumption by heterotrophic organisms and, therefore, trophic enrichment of 15N, which increases δ15N values of organic nitrogen with depth (Altabet 1988). It is this source of surface derived organic nitrogen that supplies non-vent deep-sea food webs, which exhibit relatively high δ15N values (Drazen et al. 2008; Iken et al. 2001).

Stable isotope analysis of archival tissues, or tissue that have little or no elemental turnover once formed, can give a frozen record or these nutritional sources at the time of formation. Recently, stable isotope analysis of eye lens layers in cephalopods has been used to obtain a lifetime-long sequence of stable isotope variations, including historical changes in nutritional sources such as ontogenetic dietary changes (Hunsicker et al. 2010; Parry 2003).

Here we assess participation in the hydrothermal vent food web using the carbon and nitrogen stable isotopic composition of sequential eye lens layers of a Graneledone cf boreopacifica collected within a hydrothermal vent field. Single observations form an important component of our knowledge of behavior and diet of deep-sea and oceanic cephalopods due to a combination of difficulty of collection of cephalopods and difficulty of accessing these habitats.

Even among shallow water cephalopods low n-values, such as studies based on six individuals

89 (Petza et al. 2006)or among deep-sea and oceanic cephalopods studies based on three (Daly &

Peck 2000) or two individuals (Seibel et al. 2000) are common. Among hydrothermal vent- associated cephalopods, where collection is especially difficult, data from single individuals or observations regularly contribute important information on the life histories of these species

(González et al. 2008; Voight 2000a; Voight 2005).

METHODS Using the manned submersible DSV ALVIN (Dive #4423) one male Graneledone cf boreopacifica (mantle length = 126 mm, total length = 541 mm) was collected from Middle

Valley vent field near a chemosynthetic ecosystem at Puppy Dog vent on the Juan de Fuca Ridge

(48° 26' 52.2” N 128° 42' 16.0” W, Figure 1) at 2420 meters depth. The octopus was collected on soft sediment substrate. Immediately after collection three low-flow chemosynthetic communities were located using ALVIN near (22 m, 24 m, and 30 m) the location of the octopus.

These communities were dominated by the vestimentiferan tube worm Ridgea piscesae and also contained Paralvinella palmiformis, galatheid crabs, Lepetodrilus fucensis. The latter two species have been previous found in Graneledone cf. boreopacifica stomachs (Voight 2000a;

Voight 2012). The octopus was sacrificed by crushing the cranial cartilage with the manipulator arm and was placed in a fine mesh bag for transport to the surface. Octopuses sampled by

ALVIN often are able to escape before transport to the surface, including other octopuses that were captured during this research cruise. No animal parts were observed in the mesh bag after recovery of the octopus that would suggest evacuation of gut contents occurred during transport to the surface. Shipboard the octopus was frozen at -80°C and subsequently transported to

Pullman, WA on dry ice. In Pullman, WA the octopus was thawed and digestive track including

90 stomach was examined for contents and none were found.

For stable isotope analysis both eye lenses were collected from the Graneledone. Eye lenses were sectioned by first measuring the diameter of the eye lens with digital calipers and then peeling off of the lens layers in the outer approximately 0.5mm, hereafter referred to as a growth layer group (GLG). This process of removing successive GLGs was then repeated with the remainder of the lens until only the inner 0.2 mm remained, which was used as the final

GLG. Each GLG position in the lens was recorded as the diameter (mm) of the lens before the

GLG was removed. For instance, in a lens with a total diameter of 15 mm, the first GLG removed would be 15 mm. Each successive layer removed would correspond to the smaller diameter of the remaining lens, approaching zero toward the center. Excised GLGs were placed into separate microcentrifuge tubes and dried at 60°C for at least 24 hrs.

Dried eye lens were ground to a fine powder and approximately 0.50 mg was placed into a tin capsule for isotopic analysis of 13C/12C and 15N/14N ratios. Samples were analyzed using a

13 15 Belemnite for carbon and atmospheric N2 for nitrogen. Routine precision for δ C and δ N was

+/- 0.1 ‰ and 0.3 ‰ respectively.

For comparison to Graneledone eye lens stable isotope values for Juan de Fuca Ridge hydrothermal vent fauna and northeast Pacific abyssal fauna were taken from literature sources

(Bergquist et al. 2007; Drazen et al. 2008; Levesque et al. 2003; Levin & Michener 2002; Limén et al. 2007; McKiness et al. 2005; Van Dover & Fry 1994). Species averages were taken from

91 tables or extracted from graphs using g3data software (Bauer & Reynolds 2008)(see

Supplemental Table 2 for complete dataset).

Possible mixing solutions for vent and abyssal inputs represented in Graneledone eye lens stable isotope composition was determined using the bayesian stable isotope mixing model in the IsotopeR package in R (Hopkins & Ferguson 2012). Model was run using 3 markov chain monte carlo chains running for 75,000 iterations with 10,000 iteration burnin. Trophic enrichment factors for δ15N and δ13C were assumed to be 3.3 ± 0.5 ‰ and 0 ± 0.75 ‰, respectively (Hobson & Cherel 2006). Mixing solutions were determined at each eye lens GLG.

RESULTS Graneledone cf boreopacifica raw eye lens δ15N values varied between 21 ‰ to 16.5 ‰, the former being the highest δ15N value reported for any fauna closely associated with the chemosynthetic ecosystems of the Juan de Fuca Ridge vent system. This also represented a 4.5‰ ontogenetic decrease in δ15N values of eye lens layers within the inner half of the lens, one of the largest ontogenetic decreases ever reported for an individual cephalopod (Hunsicker et al. 2010;

Lorrain et al. 2011; Parry 2003; Ruiz-Cooley et al. 2010). Isotopic composition of arm and mantle muscle (-18.0 ± 0.5 ‰ δ13C, 16.6 ± 0.6 ‰ δ15N) closely corresponded to isotopic composition of the outermost eye lens (-18.9 ‰ δ13C, 17.0 ‰ δ15N, Fig 7). Graneledone δ15N values were highest at the beginning of life, and consistently declined for the first half of the eye lens before settling around 16.5 ‰ for the remainder of the lens. Graneledone eye lens δ13C values ranged from -19.3 ‰ to -17.5 ‰ with values following a similar pattern to nitrogen.

Carbon and trophic enrichment corrected nitrogen stable isotope values (-3.3 ‰ δ15N) of

Graneledone eye lens layers fall well within the variation observed for fauna from nearby non-

92 vent deep-sea food webs (Fig 7). The lowest Graneledone eye lens trophic enrichment corrected

δ15N (13.2 ‰) values were well above even the most 15N enriched vent fauna reported in the literature from the Juan de Fuca vent system, a crab from Axial Volcano with a δ15N of 11.5‰.

The vent fauna considered here includes all vent organisms that have been identified from

Graneledone guts (Voight 2000a), and many additional common vent organisms including vent- endemic bivalves that have been hypothesized to fall prey to Graneledone (Juniper et al. 1992;

Mottl et al. 1998).

The IsotopeR mixing model, however, reveals there are mixing solutions that do incorporate a small amount of vent fauna into the diet (Fig 8A). Mean mixing solutions include

5 % to 8 % contribution from vent fauna depending on eye lens position, however more than a quarter of mixing solutions at all eye lens positions include no vent contribution. There is a increase in the mean mixing solution vent fauna dietary contribution over the first half of the life of the octopus, reflecting the decrease in eye lens δ15N values (Fig 8B). Nevertheless, eye lens isotopic composition suggests that the Graneledone cf. boreopacifica examined here consumed very little, if any, vent fauna over the duration of its life.

DISCUSSION The trophic ecology of deep-sea octopuses is an area of study that has been hampered by difficult access to the habitat and the difficultly in observing or capturing the active, mobile targets, resulting in little data. Here stable isotope analysis of an archival tissue, eye lens layers, maximizes the amount of data that can be collected from a single individual, revealing dietary contributions throughout the life of the animal. It is conceivable that the single organism investigated here is not representative of the Graneledone cf. boreopacifica found in association

93 with hydrothermal vents. Even so, single individuals represent significant additions to the literature on the trophic ecology of this species and this genus, especially in this case in which the application of a novel method has given a life-long view of the diet of this individual.

Collection of octopuses near hydrothermal vent habitats requires use of submersibles (Voight &

Drazen 2004), unlike collection in abyssal plain or pelagic habitats that can be accomplished by trawl (Quetglas et al. 2001). This added difficulty of access makes even single collections or observations of these organisms in situ potentially large contributions to the trophic ecology of this group (Voight 2000a; Voight 2005).

Graneledone cf. boreopacifica has been observed aggregating near hydrothermal vents in the northeastern Pacific (Drazen et al. 2003; Voight 2000b). These observations have lead to the hypothesis that these octopuses are attracted to vent communities to take advantage of abundant prey (Juniper et al. 1992; Mottl et al. 1998). The Graneledone cf boreopacifica investigated here does not appear to have included vent fauna as a large portion of its diet for any period of time during its life. This is despite being collected within 30 m of productive chemosynthetic habitats containing prey items found in the gut contents of this species, such as the limpet Lepetodrilus fucensis and galatheid crabs. Previous investigations have found a Graneledone cf boreopacifica in the Juan de Fuca Ridge vent system with considerable amounts of vent-endemic organisms in the gut, including 27 of the gastropod Provanna variabilis, three Lepetodrilus fucensis, three

Nereis piscesae and 43 polynoid polychaetes (Voight 2000a). This demonstrates that octopuses of this genus will, at least occasionally, consume vent fauna. However, this study demonstrates that although Graneledone cf. boreopacifica may occur in close association with vent habitats, and although able to consume vent organisms, may still, paradoxically, not be utilizing these highly

94 productive habitats to contribute to their diet.

It may be that Graneledone are only weakly tolerant of the high levels of sulfides present in vent environments and fauna. Sulfide is very toxic and abundant in hydrothermal vent habitats and vent-endemic organisms. Sulfide disrupts the activity of cytochrome c oxidase, denatures proteins by reducing disulfide bridges, and binds to heme rings (Grieshaber & Völkel

1998; Wang & Chapman 2009). There is no direct evidence that Graneledone cf. boreopacifica has any physiological mechanisms to deal with sulfide, and has been shown to lack major reservoirs of hypotaurine (Yancey et al. 2002), an osmolyte commonly used by diverse marine invertebrates to detoxify high levels of sulfide (Ortega et al. 2008; Rosenberg et al. 2006). It may be that octopuses are only able to consume vent fauna in short bursts, or at low consistent levels, while keeping the majority of the diet composed of alternative food sources.

Alternatively, Graneledone, may aggregate near hydrothermal vents to reproduce where

Graneledone females can take advantage of the solid substrates, which are rare in the deep ocean, are available in active and dead vent fields, and appear to be required to lay and brood eggs (Drazen et al. 2003; Voight & Grehan 2000). Aggregation of males and females near solid substrate may not only provide required egg-laying habitat, but also help ameliorate one of the principle challenges of deep-sea organisms: finding an appropriate mate when population levels are low in comparison to available habitat (Baird & Jumper 1995).

Away from hydrothermal vents on sediment substrate Graneledone have been observed extending the distal half of each arm into the sediment (Voight 2008). This has been interpreted as foraging for infaunal organisms and, while no direct observation of prey capture using this method have been observed, slight movements of the arm toward the mouth seem in indicate the

95 passage of material to the mouth along successive suckers (Voight 2008). Graneledone cf boreopacifica may continue to prey on abyssal infaunal prey if seafloor sediments are available when aggregating near hydrothermal vents to breed, and only switch to foraging in vent habitats if appropriate sediments are unavailable in the local environment.

This hypothesis would predict that hydrothermal vent-endemic fauna in the diet of vent- associated Graneledone cf boreopacifica would show a positive correlation with the percent solid substrate in the local environment. The data presented here would support this hypothesis:

Middle Valley, where the Graneledone reported on here was collected, has abundant seafloor sediments in which octopuses may forage for sediment infauna immediately adjacent to the vents

(Grehan & Juniper 1996). Axial volcano, however, is an active volcano that has experienced recent eruptions in 2011 and 1998 producing new basalt, and has a caldera with a high proportion of solid, rather than sediment, substrate (Caress et al. 2012). It was from this site that a

Graneledone was found to have consumed many vent-endemic gastropods and polychaetes

(Voight 2000a).

Additionally, the decrease in δ15N values over the first third of life as revealed by eye lens stable isotope analysis, which then holds relatively constant for the final half of the octopus' life

(Fig. 7), also fits this hypothesis. Most marine predators show an ontogenetic increase in δ15N values with increase in size corresponding with consuming prey from higher trophic levels

(Cocheret de la Morinière et al. 2003; Estrada et al. 2006). One scenario that is consistent with this ontogenetic decrease in δ15N values is movement by the octopus to the vent system relatively early in life and after which a low amount of vent-derived nutrition is consumed by the octopus for the remainder of its life (Fig 8). This would not necessarily be a result of consuming

96 organisms that are a part of the chemosynthetic system. Non-vent deep-sea organisms near chemosynthetic ecosystems may still bear some chemosynthetic stable isotope signature

(MacAvoy et al. 2008) and consuming non-vent fauna immediately adjacent to vents could lead to a low degree of incorporation of vent-derived nutrients into the octopus' diet.

It is possible that the octopus reported on here moved only recently into the hydrothermal vent habitat. However, without an ontogenetic move into the vent system much earlier, near midlife, it would difficult to explain a large (4.5‰) drop in δ15N values. Also, brooding of

Graneledone eggs is estimated to take as long as four years (Voight & Grehan 2000), and females appear to spawn with multiple males (Bello 2006). Such drawn out periods of reproductive activity mean that Graneledone likely remain in habitat conducive to reproduction for extended periods of time. If this is true, any octopus taken from reproductive grounds will have likely been in the area for more than a short time.

It is unknown how far Graneledone travel in search of food, however it is likely that travel is quite slow relative to other cephalopods. Graneledone are more reluctant to swim away from submersibles than co-occurring Benthoctopus species (Voight 2008), and appear to have a metabolic rate lower than other octopods (Seibel & Childress 2000). Beyond the Puppy Dog vent the next nearest vent in the Middle Valley vent field is Inspired Mounds, approximately 834 m away from the octopus collection point, and the nearest known vent field beyond Middle

Valley is the Sasquatch vent field on the Endeavor segment approximately 57 km away. While travel between vents in Middle Valley would appear possible, immigration of this octopus from another vent field does not seem likely.

In conclusion, this work demonstrates that although Graneledone cf boreopacifica may

97 be often found near highly productive hydrothermal vent habitats, and appear to be able to consume the fauna located in those habitats, those individuals do not necessarily consume large amounts vent-endemic fauna. As a result of this finding I hypothesize that Graneledone cf. boreopacifica may primarily aggregate near hydrothermal vent areas for reproduction and forage on non-vent infaunal organisms unless appropriate sediments are not available. Only if appropriate sediments are not available in the local area, therefore, would these octopuses begin to consume vent-endemic organisms.

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105 Figure 7: Map of Graneledone collection location in the Dead Dog vent field of Middle Valley on the Juan de Fuca Ridge vent system (left), and geographic location of Middle Valley, and locations of collection points of literature obtained potential prey items (right). Black lines in right map indicate tectonic plate margins.

106 Figure 8: Graneledone cf boreopacifica eye lens layer carbon and nitrogen stable isotope values (gray circles) plotted versus isotopic values of potential prey items from vent (black circles) and abyssal (white circles) food webs. Vent genera that has been found in Graneledone cf boreopacifica guts are marked with a white “x”. Graneledone eye lens value circles have been scaled proportionally to eye lens diameter at a given layer, thus large circles indicate layers for later in life. Graneledone eye lens δ15N values were reduced by 3.3 ‰ to correct for trophic enrichment of 15N. Stable isotope composition of muscle taken from both arm and mantle is shown as dark grey circle and marked (mean ± SD, also reduced by 3.3 ‰ δ15N).

107 Figure 9: Proportion composition of hydrothermal vent fauna (black) and abysal fauna (gray) in the lifetime diet of a Graneledone cf boreopacifica collected from Middle Valley, Juan de Fuca Ridge vent system as determined using a bayesian stable isotope mixing model (IsotopeR) from carbon and nitrogen stable isotope compositions of sequential eye lens layers. White dashed line indicates lower 95% confidence interval; upper 95% CI is 1 at all lens diameters and is thus not shown. Figure B shows expanded view of proportions 0.91 to 0.96 to better show trend.

108 Supplemental Table 2. Literature stable isotopic values from vent and abyssal fauna used for comparison to Graneledone eye lens.

Species Habitat Site δ13C δ15N Reference Hydroids vent Endeavor −22.1 2.9 Van Dover & Fry, 1994 Paralvinella vent Endeavor −25.2 4.1 Van Dover & Fry, 1994 Amphisamytha vent Endeavor −20.7 5.6 Van Dover & Fry, 1994 Polynoid vent Endeavor −20.6 5.8 Van Dover & Fry, 1994 Nicomache vent Endeavor −26.2 4.2 Van Dover & Fry, 1994 Lepetodrilus fucensis vent Endeavor −25.6 1.3 Van Dover & Fry, 1994 Buccinum viridum vent Endeavor −17.3 2.9 Van Dover & Fry, 1994 Ridgeia piscesae vent Endeavor −14.6 1.8 Van Dover & Fry, 1994 Paralvinella palmiformis vent Axial Volcano −10.1 7.4 Levesque et al, 2003 Paralvinella palmiformis vent Endeavor −21.8 2.0 Levesque et al, 2003 Paralvinella palmiformis vent Endeavor −16.7 −1.6 Levesque et al, 2003 Paralvinella palmiformis vent Axial Volcano −12.5 6.2 Levesque et al, 2003 Paralvinella palmiformis vent Axial Volcano −10.1 6.9 Levesque et al, 2003 Paralvinella palmiformis vent Axial Volcano −9.8 6.5 Levesque et al, 2003 Paralvinella palmiformis vent Axial Volcano −11.2 5.4 Levesque et al, 2003 Paralvinella palmiformis vent Axial Volcano −9.2 7.6 Levesque et al, 2003 Paralvinella palmiformis vent Axial Volcano −11.1 6.9 Levesque et al, 2003 Paralvinella sulfincola vent Axial Volcano −12.0 6.6 Levesque et al, 2003 Paralvinella sulfincola vent Endeavor −19.5 1.2 Levesque et al, 2003 Paralvinella sulfincola vent Endeavor −18.7 1.0 Levesque et al, 2003 Paralvinella sulfincola vent Axial Volcano −13.9 8.0 Levesque et al, 2003 Paralvinella sulfincola vent Axial Volcano −11.8 7.4 Levesque et al, 2003 Paralvinella sulfincola vent Axial Volcano −9.8 9.5 Levesque et al, 2003 Paralvinella pandorae vent Axial Volcano −8.7 6.7 Levesque et al, 2003 Paralvinella pandorae vent Axial Volcano −10.3 6.2 Levesque et al, 2003 Paralvinella pandorae vent Axial Volcano −10.8 5.6 Levesque et al, 2003 Paralvinella pandorae vent Axial Volcano −8.6 8.0 Levesque et al, 2003 Paralvinella pandorae vent Axial Volcano −8.7 7.3 Levesque et al, 2003 Coryphaenoides yaquinae abyss Station M −17.6 18.5 Drazen et al, 2008 Coryphaenoides armatus abyss Station M −17.4 18.1 Drazen et al, 2008 Travisia abyss Station M −16.8 19.7 Drazen et al, 2008 Caridean Shrimp abyss Station M −16.9 19.5 Drazen et al, 2008 Laetmonice abyss Station M −17.0 18.5 Drazen et al, 2008

109 Munidopsis abyss Station M −18.2 18.0 Drazen et al, 2008 Protankyra brychia abyss Station M −18.1 17.3 Drazen et al, 2008 Lysianassid amphipods abyss Station M −21.7 17.4 Drazen et al, 2008 Bathyphellia australis abyss Station M −17.5 16.5 Drazen et al, 2008 Ophiacantha abyss Station M −19.5 15.4 Drazen et al, 2008 Ophiura bathybia abyss Station M −20.0 15.2 Drazen et al, 2008 Ophiura mutabilis abyss Station M −19.9 14.9 Drazen et al, 2008 Paradiopatra abyss Station M −20.5 14.2 Drazen et al, 2008 Abyssocucumis abyssorum abyss Station M −19.8 14.4 Drazen et al, 2008 Peniagone vitrea abyss Station M −19.5 14.1 Drazen et al, 2008 Paralvinella sulfincola vent Axial Volcano −9.7 6.3 Limén et al, 2007 Paralvinella palmiformis vent Axial Volcano −10.0 5.8 Limén et al, 2007 Lepetodrilus fucensis vent Axial Volcano −13.8 5.2 Limén et al, 2007 Depressogyra globulus vent Axial Volcano −14.2 7.1 Limén et al, 2007 S. quadrispinosus vent Axial Volcano −13.2 4.8 Limén et al, 2007 Ridgea piscesae vent Axial Volcano −11.0 −0.1 Limén et al, 2007 Amphisamytha galapagensis vent Axial Volcano −17.4 6.6 Limén et al, 2007 Polynoid vent Axial Volcano −15.6 8.2 Limén et al, 2007 Lepetodrilus fucensis vent Axial Volcano −13.9 6.9 Limén et al, 2007 Depressogyra globulus vent Axial Volcano −15.0 4.8 Limén et al, 2007 Buccinum viridum vent Axial Volcano −18.8 6.4 Limén et al, 2007 Buccinum viridum vent Axial Volcano −22.9 5.7 Limén et al, 2007 Euphilomedes climax vent Axial Volcano −18.7 8.4 Limén et al, 2007 Ridgeia piscesae vent Axial Volcano −11.1 2.9 Limén et al, 2007 Juv. Crab vent Axial Volcano −22.0 11.5 Limén et al, 2007 Nereis vent Axial Volcano −17.1 9.8 Limén et al, 2007 Nereis juv vent Axial Volcano −21.0 9.7 Limén et al, 2007 Amphisamytha galapagensis vent Axial Volcano −20.8 7.1 Limén et al, 2007 Ammothea verenae vent Axial Volcano −19.0 8.6 Limén et al, 2007 Lepetodrilus fucensis vent Axial Volcano −15.4 7.0 Limén et al, 2007 Depressogyra globulus vent Axial Volcano −24.4 7.5 Limén et al, 2007 Buccinum viridum vent Axial Volcano −21.0 7.6 Limén et al, 2007 Buccinum viridum vent Axial Volcano −20.9 5.7 Limén et al, 2007 Buccinum viridum vent Axial Volcano −24.4 7.1 Limén et al, 2007 Euphilomedes climax vent Axial Volcano −17.5 7.4 Limén et al, 2007 Copidognathus papillatus vent Axial Volcano −21.7 7.9 Limén et al, 2007 Paracanthonchus vent Axial Volcano −21.7 8.7 Limén et al, 2007

110 Ridgeia piscesae vent Axial Volcano −11.1 1.0 Limén et al, 2007 Ridgeia piscesae vent Endeavor −14.5 1.5 Bergquist et al, 2007 Nicomache vent Endeavor −18.8 1.2 Bergquist et al, 2007 Ophryotrocha globopalpata vent Endeavor −25.0 −5.6 Bergquist et al, 2007 Scoloplos vent Endeavor −25.1 3.2 Bergquist et al, 2007 Hesiospina vestimentifera vent Endeavor −23.3 −2.3 Bergquist et al, 2007 Protomystides verenae vent Endeavor −17.4 3.8 Bergquist et al, 2007 Branchinotogluma grasslei vent Endeavor −15.8 4.8 Bergquist et al, 2007 Branchinotogluma hessleri vent Endeavor −15.5 5.2 Bergquist et al, 2007 Branchinotogluma sandersi vent Endeavor −18.1 5.2 Bergquist et al, 2007 Opisthotrochopodus tunnicliffeae vent Endeavor −15.4 4.7 Bergquist et al, 2007 Lepidonotopodium piscesae vent Endeavor −14.9 3.1 Bergquist et al, 2007 Lepidonotopodium williamsae vent Endeavor −18.2 4.5 Bergquist et al, 2007 Levensteiniella intermedia vent Endeavor −23.2 6.8 Bergquist et al, 2007 Levensteiniella kincaidi vent Endeavor −18.9 7.1 Bergquist et al, 2007 Neoleanira racemosa vent Endeavor −24.9 7.8 Bergquist et al, 2007 Sphaerosyllis ridgensis vent Endeavor −23.6 7.7 Bergquist et al, 2007 Paralvinella pandorae vent Endeavor −22.0 −4.7 Bergquist et al, 2007 Paralvinella palmifornis vent Endeavor −22.7 −3.9 Bergquist et al, 2007 Amphisamytha galapagensis vent Endeavor −21.9 5.6 Bergquist et al, 2007 Helicoradomenia juani vent Endeavor −17.5 5.3 Bergquist et al, 2007 Buccinum viridum vent Endeavor −19.8 3.8 Bergquist et al, 2007 Provanna variabilis vent Endeavor −28.5 −0.6 Bergquist et al, 2007 Depressigyra globulus vent Endeavor −18.0 1.1 Bergquist et al, 2007 Clypeosectus curvis vent Endeavor −32.0 6.8 Bergquist et al, 2007 Lepetodrilus fucensis vent Endeavor −23.1 1.2 Bergquist et al, 2007 Idas washingtonia vent Endeavor −30.7 1.5 Bergquist et al, 2007 Copidognathus papillatus vent Endeavor −23.6 0.1 Bergquist et al, 2007 Euphilomedes climax vent Endeavor −23.3 1.3 Bergquist et al, 2007 Seba profundus vent Endeavor −21.2 5.1 Bergquist et al, 2007 Ammothea verenae vent Endeavor −22.6 1.7 Bergquist et al, 2007 Sericosura venticola vent Endeavor −24.1 6.6 Bergquist et al, 2007 Sericosura ditta vent Endeavor −21.2 4.0 Bergquist et al, 2007 Nematode vent Endeavor −20.4 6.3 Bergquist et al, 2007 Nemertean vent Endeavor −23.7 9.0 Bergquist et al, 2007 Ciliophora vent Endeavor −32.3 3.8 Bergquist et al, 2007 Allocentrotus fragilis abyss Oregon Margin −23.1 10.8 Levin & Michener, 2002

111 Allocentrotus fragilis abyss Oregon Margin −23.3 12.0 Levin & Michener, 2002 macroinfaunal sp. abyss Oregon Margin −24.0 15.4 Levin & Michener, 2002 macroinfaunal sp. abyss Oregon Margin −21.5 11.4 Levin & Michener, 2002 macroinfaunal sp. abyss Oregon Margin −20.9 11.8 Levin & Michener, 2002 macroinfaunal sp. abyss Oregon Margin −20.7 11.0 Levin & Michener, 2002 Chionoecetes tanneri abyss Oregon Margin −19.1 15.0 Levin & Michener, 2002 Chionoecetes tanneri abyss Oregon Margin −18.3 14.7 Levin & Michener, 2002 macroinfaunal sp. abyss Oregon Margin −17.2 16.1 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −24.2 8.7 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −21.7 9.2 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −22.0 12.6 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −19.6 9.1 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −20.1 11.9 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −18.2 12.0 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −18.1 12.6 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −18.2 12.6 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −17.5 13.1 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −17.6 12.2 Levin & Michener, 2002 macroinfaunal sp. abyss Eel River Margin −16.2 14.4 Levin & Michener, 2002 Bathymodiolus sp. vent Endeavor -26.6 5.19 McKiness et al, 2005

112