Author’s Accepted Manuscript

Demographic indicators of change in a deposit- feeding abyssal holothurian community (station M, 4000m)

Christine L. Huffard, Linda A. Kuhnz, Larissa Lemon, Alana D. Sherman, Kenneth L. Smith

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PII: S0967-0637(15)30099-6 DOI: http://dx.doi.org/10.1016/j.dsr.2016.01.002 Reference: DSRI2569 To appear in: Deep-Sea Research Part I Received date: 25 August 2015 Revised date: 9 January 2016 Accepted date: 9 January 2016 Cite this article as: Christine L. Huffard, Linda A. Kuhnz, Larissa Lemon, Alana D. Sherman and Kenneth L. Smith, Demographic indicators of change in a deposit-feeding abyssal holothurian community (station M, 4000m), Deep-Sea Research Part I, http://dx.doi.org/10.1016/j.dsr.2016.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1

Demographic indicators of change in a deposit-feeding abyssal holothurian community (Station M, 4000 m)

Christine L. Huffard a* , Linda A. Kuhnz a, Larissa Lemon a,b , Alana D. Sherman a, Kenneth L. Smith, Jr. a aMonterey Bay Aquarium Research Institute. Moss Landing, CA bCalifornia State University Monterey Bay. Seaside, CA

*Corresponding author: [email protected]. Monterey Bay Aquarium Research Institute, 7700 Sandholdt Rd, Moss Landing, CA 95039

Abstract

Holothurians are among the most abundant benthic megafauna at abyssal depths, and important consumers and bioturbators of organic carbon on the sea floor. Significant fluctuations in abyssal holothurian density are often attributed to species-specific responses to variable particulate organic carbon flux (food supply) stemming from surface ocean events. We report changes in densities of 19 holothurian species at the abyssal monitoring site Station M in the northeast Pacific, recorded during 11 remotely operated vehicle surveys between Dec 2006 and Oct 2014. Body size demographics are presented for Abyssocucumis abyssorum, Synallactidae sp. 1, Paelopatides confundens , Elpidia sp. A, Peniagone gracilis , Peniagone papillata , Peniagone vitrea , Peniagone sp. A, Peniagone sp. 1, and Scotoplanes globosa . Densities were lower and species evenness was higher from 2006 –2009 compared to 2011 – 2014. Food supply of freshly-settled phytodetritus was exceptionally high during this latter period. Based on relationships between median body length and density, numerous immigration and juvenile recruitment events of multiple species appeared to take place between 2011 and 2014. These patterns were dominated by elpidiids (Holothuroidea: Elasipodida: Elpidiidae), which consistently increased in density during a period of high food availability, while other groups showed inconsistent responses. We considered minimum body length to be a proxy for size at juvenile recruitment. Patterns in density clustered by this measure, which was a stronger predictor of maximum density than median and mean body length.

Keywords

Ecology, Population biology, Deep-sea, Echinodermata, Spawning aggregation, Body size

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1. Introduction

The deep sea offers unique model systems for studying patterns and mechanisms associated with community change. The co-existence of different species utilizing overlapping resources within the same habitat, and changes in their relative abundance over time, have been a long-standing interest of ecologists (Pianka, 1973; Menge and Sutherland, 1976). However, many key ecological variables associated with community composition change in traditionally studied ecosystems (e.g. terrestrial or shallow-water marine) are either significantly different in, or absent from, the deep sea. For example photoperiod, temperature, and salinity are common determinants of local distribution (Ricketts et al. , 1992) and reproductive period in shallow water invertebrates (Giese, 1959) including holothurians (McEuen, 1988; Babcock et al. , 1992), but these factors do not vary considerably at abyssal depths (4,000 –6,000 m deep; Gage and Tyler, 1991). By contrast food supply, in the form of sinking particulate organic carbon, can be highly limited and variable in the deep sea. Periods of undersupply can last years to decades, and the magnitude of episodic inputs often vary more inter-annually than seasonally (Lampitt et al. , 2010; Smith et al. , 2013). Benthic organisms living both within and on top of the sediment increase in abundance following food pulses (Pfannkuche et al. , 1999; Galéron et al. , 2001). As a result, the relative access to high quality food appears to be a key driver of community composition over time at abyssal depths (Sibuet, 1985; Gage and Tyler, 1991; Gooday, 2002; Ruhl and Smith Jr, 2004; Baillon et al. , 2011; Gambi et al. , 2014). In terms of abundance and biomass, holothurians (Echinodermata: Holothuroidea) are often the dominant mobile megafauna group on the abyssal sea floor [(Gage and Tyler, 1991; Hansen, 1975; Lauerman et al. , 1996; Ohta, 1983; Ruhl, 2007; Sibuet, 1985) but see (Durden et al. , 2015a); Megafauna are defined as those animals large enough to be visible in photographs (Grassle et al. , 1975)]. They are major consumers and bioturbators of organic carbon (Ginger et al. , 2001), presumably feeding as they transit an area (Bett et al. , 2001). For the entire megabenthic community, seafloor reworking rates have been estimated on the order of one to two and a half years (Bett et al. , 2001; Kaufmann and Smith, 1997), but as rapid as six weeks during a holothurian “boom” event (Bett et al. , 2001). Surface and sub-surface deposit feeding holothurians can account for a majority of reworking activity, in some cases out-pacing other taxonomic assemblages by a factor of 9:1 (Kaufmann and Smith, 1997). When present in high densities holothurians can rapidly deplete the quantity and quality of organic material in abyssal surface sediments (Bett et al. , 2001; Ginger et al. , 2001). Through consumption and repackaging, they broadly influence the availability and composition of food for other organisms (Billett et al. , 2010; MacTavish et al. , 2012), which can lead to cascading impacts on subsurface deposit feeders (Iken et al. , 2001), infauna 3

(Soto et al. , 2010; Thistle et al. , 2008), and scavenging fishes (Bailey et al. , 2006). Abrupt, large-scale changes in holothurian abundances have corresponded with ecological shifts impacting multiple phyla that have lasted years to possibly a decade (Billett et al. , 2010; Gooday et al. , 2010; Kuhnz et al. , 2014; Ruhl, 2007; Ruhl and Smith Jr, 2004; Wigham et al. , 2003a). Shifts in abyssal holothurian communities have been traced to surface processes (Smith et al. , 2008), making them potential indicators of climate change impacts on deep-sea ecosystems and carbon remineralization processes (Glover et al. , 2010). In a habitat that can experience chronic food limitation punctuated by episodic pulses of essential food, those holothurian species that are more efficiently able to locate and utilize fresh phytodetritus are proposed to gain a competitive advantage over those that are restricted by slower locomotion, non- selective feeding tentacle morphology, and/or other physiological limitations (Hudson et al. , 2003; Iken et al. , 2001; Neto et al. , 2006; Wigham et al. , 2003a). Abyssal holothurians consume freshly-settled phytodetritus when available, and some species might require surface-derived phytopigments and sterols for reproduction (Drazen et al. , 2008; FitzGeorge-Balfour et al. , 2010; Ginger et al. , 2001; Iken et al. , 2001; Lauerman and Kaufmann, 1998). These nutrients can be limiting in the deep-sea (Hudson et al. , 2003; Wigham et al. , 2003a). Pulses of phytodetritus to the sea floor appear to trigger opportunistic reproductive activity in numerous deep-sea echinoderms including holothurians (Tyler et al . 1982; Eckelbarger and Watling, 1995; Galley et al. , 2008; Wigham et al. , 2003b). High-flux events represent especially favorable conditions because organic material reaching the sea floor can maintain a high fraction of its surface-derived nutrients (Conte et al. , 1998). Highly mobile surface deposit feeding holothurians quickly utilize fresher surface material, leaving larger, slower subsurface deposit feeders to consume more degraded forms (Iken et al. , 2001). In some cases grazing rates by a few holothurian species together can match deposition rates of fresh material, thereby monopolizing a limiting resource (Bett et al. , 2001; Wigham et al. , 2003a). This differential access to food can lead to interspecific differences in reproductive effort and ultimately abundance (Ramirez-Llodra et al. , 2005). In recent years the supply of surface-derived food, including phytodetritus, at the abyssal time series monitoring site Station M (northeast Pacific, ~4000m) has exhibited high magnitude episodic pulses relative to 1989 –2009 (Smith et al. , 2014). The first two decades of the time-series were dominated by periods of food undersupply (Smith et al. , 2013). Corresponding with recent increases in food input, the proportional density of holothurians in relation to other megafauna groups also increased, from less than 5% in early 2007 to nearly 50% by mid-2011; mobile surface deposit feeders (primarily holothurians) gained numerical dominance over sessile suspension feeders, signifying a major shift in the megafauna community (Kuhnz et al. , 2014). Given the known and suspected links between food supply and deep-sea holothurian reproduction, and the exceptionally high magnitudes of fresh phytodetritus influx observed recently at 4

Sta. M, we hypothesized recent food oversupplies might have fueled broad-scale changes in holothurian communities. We compared holothurian demographics at Sta. M from 2006 –2009 and 2011 –2014 to better understand species-specific responses during the megafauna community transition described by Kuhnz et al . (2014). Using video collected by remotely operated vehicles (ROV) we assessed changes in density (19 species) and body length (BL) distributions (10 species), factors which are often used to detect reproductive recruitment to the sea floor and immigration events (Billett et al . 2001; Ramirez- Llodra et al. , 2005; Ruhl, 2007; Kalogeropoulou et al. , 2010). Body size is associated with numerous ecological factors including population density and reproductive potential (Pianka, 1974). Species with small body size characteristics and/or high fecundity are predicted to gain ecological advantages in colonizing unpredictable resources (Pianka, 1974) including at abyssal depths (Billett and Hansen 1982; Billett 1991). Our dataset allowed us to test whether aspects related to body size were associated with the ability to attain very high densities. Therefore we interpreted changes in holothurian densities in light of their body lengths.

Specifically, we tested the following non-mutually exclusive hypotheses: 1. Holothurian densities were higher from 2011 –2014 than 2006 –2009. 2. Immigration and juvenile recruitment events involving one or more species were more common from 2011 –2014 than 2006 –2009. 3. Body size characteristics (mean, median and minimum body length) correlated with patterns in density. A. Maximum density correlated with mean, median and minimum body length. B. Density patterns over all sampling periods clustered with the body size characteristic that best predicted maximum density. 4. Holothurian communities differed from 2006–2009 compared to 2011 –2014 as evidenced by differences in: A. measures of species diversity and evenness B. community structure.

2. Methods

2.1. Study site and instrumentation

At Station M (34° 50' N, 123° 00' W) surface water productivity corresponds with lagged food supply and megafauna responses on the abyssal sea floor [~ 4000m; (Ruhl and Smith Jr, 2004)]. The sea bed is soft sediment (silty-clay), with topographic relief <100 m over 770 km 2 (Kaufmann and Smith, 1997). Food falls as particulate organic carbon and detrital aggregates comprised primarily of 5

phytodetritus but occasionally included gelatinous material (Smith et al. , 1998; Smith et al. , 2014). Settling location of food on the sea floor at this station can be patchy (Lauerman et al. , 1996).

Between Dec 2006 and Oct 2014, two remotely operated vehicles (ROV Tiburon and ROV Doc Ricketts ) video-recorded (high-definition Ikegama video cameras fitted with HA10Xt.2 Fujinon lenses) benthic transects at Sta. M (220 –1830 m long; n = 11 survey periods; Table 1). Intervals between sampling periods were not uniform. Two parallel lasers, 29 cm apart, were projected onto the sea floor continuously during ROV transect recording, and used as a scale reference for size measurements of holothurians. The ROV imaging system yielded the ability to see and measure objects at least 3 mm in largest dimension. This resolution is sufficient for detecting specimens at the upper size range known for pelagic juveniles of species within Elasipodida, the most abundant and diverse group of deep-sea holothurians (Hansen, 1975; Tyler et al. , 1985a; Gebruk et al. , 1997). This method reduces the sampling bias of trawling, in which small individuals can be undersampled (Billett et al. , 2001; Durden et al. , 2015b), and overall abundances can be underestimated by up to 50% (Billett, 1991). However this approach is not likely to detect species that bury into the sediment. While identifications to the species level can be difficult at very small sizes, photographic survey methods are nevertheless preferred over trawls for assessing sizes and densities of deep-sea holothurians (Billett, 1991; Bluhm and Gebruk, 1999; Rogacheva and Gebruk, 2013; Durden et al. , 2015b).

2.2. Animal identification and sizing from ROV footage

ROV transect video footage was annotated in the Monterey Bay Aquarium Research Institute’s open source Video Annotation Reference System (VARS) (Schlining and Stout, 2006) for animal identification and sizing. Each holothurian in the ROV transects was identified to the lowest possible taxon [http://dsg.mbari.org/dsg/images/concept/Holothuroidea, (Jacobsen Stout et al. , 2015)] and sized following Ruhl (2007). Nineteen distinct holothurian morphotypes (referred to here as distinct species for simplicity) were distinguished, counted, and sized in ROV footage. Peniagone papillata Hansen 1975, Peniagone gracilis (Ludwig 1894) and Peniagone sp. A are visually similar and could be mistaken for each other when very small (> ~ 2 cm). Likewise for Peniagone vitrea Théel 1882 and Peniagone sp. 1.

Using the measurement tool in VARS, holothurian size (BL) was measured digitally as the distance from the anterior end (between the base of the dorsal papillae if present) to the posterior end of the holothurian, excluding feeding tentacles (Ruhl, 2007). Some Peniagone spp. postured with the anterior end raised off the seafloor. Our measurements were based on a straight line from anterior to posterior, not including slight body curvature. These lengths were obtained only for individuals that were 6

positioned along the same horizontal plane as the laser points illuminated on the sea floor in ROV footage, and then were compared to the distance between the lasers in the same frame. This sizing methodology yields low measurement errors (< 1 mm), and tends to underestimate sizes where error occurs (Dunlop et al. , 2015). Individuals were not sized if they were positioned on top of another object or strongly contorted. In rare cases very large individuals of Scotoplanes globosa and other species were not fully visible in a single video frame, precluding sizing. Abyssocucumis abyssorum is generally epibenthic, but can bury in the sediment. Small individuals of this species sometimes crawl onto or under other objects on the sea floor, making them difficult to view and size. Thus small or buried individuals might have been undersampled. When used, terms small/smaller, and large/larger compare the relative sizes of species measured in this study, rather than body size categories which might be in use elsewhere.

2.3. Statistical analyses

2.3.1. Holothurian densities from 2006 –2009 versus 2011 –2014

Holothurian densities were calculated per species by dividing the total number of individuals (ind.) measured on each transect by the total transect area annotated for that sampling period (Table 1). Densities were then adjusted to ind. ha -1 to be consistent with other studies on holothurian density (e.g. Billett et al. , 2010). We compared densities of each species from 2011 –2014 to their densities calculated for the years 2006 –2009 respectively using Wilcoxon-Mann-Whitney test (StatXact-4.0.1). All scatter plots of densities were created using SigmaPlot 10.0.

2.3.2. Indicators of immigration and juvenile recruitment

Recruitment involves the addition of individuals to a population, whether by immigration or reproduction (Ebert, 1983). Our use of the term recruitment was restricted to recognizable individuals that could be detected on the sea floor (Ebert, 1983). Species that release brooded young [as is likely for the O. mutabilis complex at Sta. M (Hansen, 1975)], or settle as juveniles from the plankton [(as in some Elasipodids (Gebruk et al ., 1997)] might be detectable immediately upon release or settlement. Species that develop into a juvenile form after settling on the sea floor would be detected only after a post- settlement lag of unknown duration. Our use of the term recruitment refers to juvenile recruitment, as immigration events are discussed separately. 7

We prepared notched box plots of BL distributions for those species represented by at least 10 size measurements each for at least three time periods. Notched box plots offered an intuitive and rapid means for comparing data distributions including those that are skewed (McGill et al. , 1978; Krzywinski and Altman, 2014). Box plots of BL were generated using the online tool provided by (Krzywinski and Altman, 2014). Notches were defined as ±1.58 x the interquartile range (IQR), and represented the 95% confidence interval of each median. Distributions could be considered significantly different from each other if the notches in their box plots did not overlap (McGill et al. , 1978; Krzywinski and Altman, 2014). Whiskers represented data points >1.5 x IQR. This method is appropriate for describing normal distributions, or non-normal distributions based on sample sizes greater than five (McGill et al. , 1978; Krzywinski and Altman, 2014). While it can be difficult to interpret populations with multiple peaks (e.g. bimodal size distributions) using this method, such distributions were very rare in our datasets. This method allowed us greater ability to examine size distributions than histograms, which can require a minimum sample size of 30 to be considered statistically useful (Krzywinski and Altman, 2014). Letters were assigned to box plots to facilitate identification of significantly different groups based on non- overlap of notches. Letters were assigned to “letter groups” in ascending alphabetical order from the largest BL distribution (A) to the smallest distribution (E) for each species.

We identified possible immigration and juvenile recruitment events by comparing changes in median BL distributions and density over consecutive sampling periods. Median rather than average BL was chosen because this measure is appropriate for comparing skewed distributions (Krzywinski and Altman, 2014), as is common for deep-sea holothurian sizes (Tyler and Billett, 1988). As in other studies (Ramirez-Llodra et al. , 2005; Ruhl, 2007; Billett et al. , 2010; Kalogeropoulou et al. , 2010) we considered an increase in density along with a reduction of average or median body size to indicate a recent recruitment event. We suspected immigration might have occurred if density and median BL both increased from one survey to the next because 1) shifts toward smaller size distributions would not occur if densities rose due to immigration rather than recruitment of new young (small) individuals (e.g. Ruhl 2007), and 2) growth alone could explain an increase in median BL, but not an increase in density as well. We did not analyze the relationships between density and BL distribution in May 2011 because of the very large sampling gap prior to that survey.

We considered minimum body length to be a proxy for size at juvenile recruitment, the size at which juveniles could be identified transiting the sea floor. Echinoderms surveyed at this size have likely passed through post-settlement mortality (Balch and Scheilbling, 2001). This trait has been associated with survivorship, growth, and population density of other marine taxa (Cargnelli and Gross, 1996; Sogard, 1997; Searcy and Sponaugle, 2001; Gagliano et al. , 2007). 8

2.3.3. Body size and density

Using StaXact 4.0.1, we calculated Spearman Rank correlation coefficients to test whether maximum density correlated with mean, median or minimum BL. We generated a 3D scatter plot of mean, median, and minimum BL using Sigma-Plot 10.0. To examine differences in density patterns based on body size characteristics, we used Primer-E 6 (Clarke and Warwick, 2001) to perform a cluster analysis using the SIMPER routine. This test returned levels of similarity between species based on patterns in their density distributions. The Bray-Curtis dissimilarity matrix was based on square-root- transformed density data, with survey dates listed as variables and species listed as samples. The minimum BL record for each species was then mapped onto the retuned dendrogram, with color corresponding to a size-based red-blue scale. Groupings that clustered at greater than the 50% level were identified by Primer-E with colored branching. Asterisks were also added to identify species known or suspected to swim in their adult form at Sta. M based on in situ observations, designation as benthopelagic in the adult form in the literature (Miller and Pawson, 1990), or suspected (in parentheses) based on the presence of a large velum (following Rogacheva and Gebruk, 2013). While many species swim facultatively when disturbed (Rogacheva et al. 2012, Rogacheva and Gebruk, 2013) we were primarily interested in species which might swim to migrate as adults.

2.3.4. Holothurian community change

To test whether holothurian communities differed in 2011 –2014 compared to 2006 –2009, we calculated species diversity (Shannon H') and species evenness (Pielou's J') for each survey using Primer- E. We then performed the Wilcoxon-Mann-Whitney test using StatXact-4.0 to compare groups of these measures based on surveys from 2011 –2014 versus 2006 –2009. Differences in community structure were assessed using non-metric multidimensional scaling (MDS) plots prepared in Primer-E based on Bray- Curtis dissimilarity indices of square-root-transformed density data. Species were listed as variables and survey dates were listed as samples. A cluster analysis was then performed using the SIMPER routine to return levels of similarity between sampling periods based on similarities in their holothurian density distributions. Similarity contours were overlain on the MDS plot to identify sampling periods that clustered at the 40 and 50 % level.

3. Results 9

3.1. Holothurian densities from 2006 –2009 versus 2011 –2014

Density of all holothurians combined ranged from a minimum of 286 ind. ha -1 in Sep 2007 to a maximum of 23,566 ind. ha -1 in Nov 2012 (Fig. 1; Table 2). Enypniastes sp. , Psychropotes depressa (Théel, 1882), Holothuroidea sp. 5 and Holothuroidea sp. 6 were detected in only one or two sampling periods each and were not analyzed further. The remaining 15 species varied in density by 1 –4 orders of magnitude from 2006 –2014 (Fig. 1; Table 2). Only Abyssocucumis abyssorum (Théel, 1886) and Synallactidae sp. 1 were detected in all sampling periods. Densities were significantly higher from 2011 – 2014 compared to 2006 –2009 for Paelopatides confundens Théel, 1886 , Psychropotes longicauda Théel, 1882 , Elpidia sp. A , P. gracilis, P. papillata, P. vitrea, Peniagone sp. A ., Peniagone sp. 1 , Peniagone sp. 2, and S. globosa (Wilcoxon-Mann-Whitney p < 0.05) . Densities were significantly lower from 2011 – 2014 compared to 2006 –2009 for Pseudostichopus mollis Théel, 1886 (Wilcoxon-Mann-Whitney p = 0.05). Densities were not significantly different from 2011 –2014 compared to 2006 –2009 for Synallactidae sp. 1, A. abyssorum , Oneirophanta mutabilis complex, and Holothuroidea sp. 4 (Wilcoxon- Mann-Whitney p > 0.05). Doublings (or greater increases) of multiple species between sampling periods were common, particularly when comparing respective consecutive sampling periods between Feb 2009 and Nov 2012. Halving (or greater reductions) of densities between consecutive sampling periods were also common, particularly when comparing densities recorded between Nov 2012 and Apr 2014 (Table 2).

3.2. Indicators of immigration and juvenile recruitment

3.2.1. Holothurian body length distributions

Details for the 10 species that could be analyzed for BL distributions are given below, in the same order as species listed in Table 2. Notched box plots are illustrated in Fig. 2. Summary statistics for BL distributions are provided in Supplementary Table 1. Relationships between minimum, mean, and median BL are plotted in Supplementary Fig. 1.

3.2.1.1. Abyssocucumis abyssorum Abyssocucumis abyssorum densities were highest in Feb 2009 (269 ind. ha -1) and lowest in Apr 2014 (42 ind. ha -1; Fig. 1A; Fig 2). Small individuals were often found located on top of other objects or sessile organisms, such as sponge stalks. These individuals could not be measured (Dunlop et al. 2015), and as such small individuals were underrepresented in BL distribution summaries. For this reason, 10

twenty percent or more (range 21-33%) of individuals could not be sized in Dec 2006, Feb 2007, Nov 2011, Nov 2012 and Apr 2014. In Feb 2009 85% of individuals could not be sized. BL distributions were skewed to high numbers of small or unsized (typically small) individuals during the periods of relatively high density in Feb 2009, Jun 2012 and Nov 2012. BL of A. abyssorum ranged from 2.3 to 15.5 cm.

3.2.1.2. Paelopatides confundens Densities of P. confundens were highest in Oct 2014 (91 ind. ha -1), and when detected, lowest in Feb 2009 (6 ind. ha -1) (Fig. 1A; Fig 2). BL distributions were skewed toward small individuals during surveys with relatively high density in Nov 2011 and Nov 2012. The period with the highest density was represented by larger rather than smaller individuals. Paelopatides confundens BL ranged from 2.8 to 23.3 cm.

3.2.1.3. Synallactidae sp. 1 Density of Synallactidae sp. 1 was relatively high in Feb 2007 (182 ind. ha -1) and Oct 2014 (172 ind. ha -1), the latter period represented by an abundance of relatively small individuals. In May 2011 individuals were larger, and found at relatively low densities (Fig. 1A; Fig 2). The lowest density documented was 10 ind. ha -1, in Nov 2011. BL of Synallactidae sp. 1 ranged from 2.6 to 18 cm.

3.2.1.4. Elpidia sp. A Elpidia sp. A was either undetected or detected in moderate numbers of small individuals in 2007, 2008, 2009, and May 2011 (0 –1295 ind. ha -1; Fig. 1C; Fig 2). Starting in Nov 2011, BL and densities were successively higher with each sampling period until the highest density was recorded in Nov 2012 (10524 ind. ha -1), after which densities were lower with each successive sampling period. Elpidia sp. A BL ranged from 0.5 to 6.9 cm. All BL distributions were the same as or differed by one letter group compared to BL distribution of the previous sampling period. Individuals sampled in Feb 2007, Feb 2009, and May 2011 were in the smallest size grouping (C). Individuals sampled in Nov 2011 and June 2012, and Apr 2014 were in the next largest size grouping (B), and those sampled in Nov 2012 and June 2013 were in the largest size grouping (A).

3.2.1.5. Peniagone gracilis Peniagone gracilis was not detected during surveys in 2007, 2008, or 2009. Records of high densities corresponded with larger size distributions, particularly in May 2011 (907 ind. ha -1) and Jun 2012 (1150 ind. ha -1; Fig. 1C; Fig 2). The two sampling periods with a population skewed toward small size distributions (Nov 2012 and Apr 2014) corresponded with relatively low densities (337 and 375 ind. ha -1 respectively) compared to highs. The rapid change from higher density in Jun to lower density in Nov 11

2012 was accompanied by a change in BL distribution from letter group A (largest) to letter group C (second smallest). Peniagone gracilis BL ranged from 1 to 10.9 cm.

3.2.1.6. Peniagone papillata Peniagone papillata was detected in low numbers in 2007, 2008 and 2009 (0 –36 ind. ha -1). Relatively high densities were recorded in May and Nov 2011 (224, 309 ind. ha -1 respectively) before pre- 2011 levels were recorded in Apr and Oct 2014 (57, 22 ind. ha -1 respectively; Fig. 1C; Fig 2). The largest size distribution (May 2011) corresponded with the second highest density record. Body length distribution during the period with highest density (Nov 2011) was skewed toward small individuals. Peniagone papillata BL ranged from 2.5 to 19.5 cm.

3.2.1.7. Peniagone vitrea Peniagone vitrea was not detected, or was detected in low numbers, in 2007 and 2008 (0 –45 ind. ha -1; Fig. 1C; Fig 2). Moderate densities of small individuals were detected in Feb 2009 (410 ind. ha -1). Higher densities of larger individuals were recorded in May and Nov 2011, and Jun 2012 (732 –966 ind. ha -1). A high density of small individuals was detected in Nov 2012 (1683 ind. ha -1). Lower density (389 ind. ha -1) of small individuals was detected in Apr 2014. The highest density was recorded in Oct 2014 (2545 ind. ha -1), when the size demographics were large. However the very high density also included a large number of small individuals. Peniagone vitrea BL ranged from 1.2 to 12.4 cm.

3.2.1.8. Peniagone sp. A Peniagone sp. A was not detected, or detected in very low numbers, in 2007, 2008 and 2009 (0 – 19 ind. ha -1). Moderate densities of the largest size fraction were detected in May 2011 (3628 ind. ha -1; Fig. 1C; Fig 2). Densities ranged from 3628 to 5321 individuals ha -1 from May 2011 through Apr 2014, with each period represented by small BL distributions. A very large number of primarily large individuals was detected in Oct 2014 (11,627 ind. ha -1). Peniagone sp. A BL ranged from 0.9 to 14.5cm. Density in Oct 2014 was double that recorded in Apr 2014, a change that corresponded with an increase in size distribution from the smallest to the second largest.

3.2.1.9. Peniagone sp. 1 Peniagone sp. 1 was not detected in 2007 –2011 (Fig. 1C; Fig 2). It was first detected in Nov 2012, with high densities (2917 ind. ha -1) of relatively large individuals, many of which appeared to be gravid (Fig. 3). Targeted collections with the ROV confirmed the presence of gametes. Moderate densities of somewhat smaller individuals were detected in Jun 2013 (1534 ind. ha -1), and large individuals in Oct 2014 (1142 ind. ha -1). Peniagone sp. 1 was detected in very low numbers in Apr 2014 12

(7 ind. ha -1). Body length of Peniagone sp. 1 ranged from 1.5 to 9.4 cm. Each of the three periods during which Peniagone sp. 1 was detected was represented by a significantly different BL distribution.

3.2.1.10. Scotoplanes globosa Scotoplanes globosa was not detected, or detected in low numbers from 2007 –2009 (0 –6 ind. ha -1). In May 2011, a moderate density of large individuals was observed (514 ind. ha -1; Fig. 1C; Fig 2). Successively higher densities were recorded during each sampling period until the highest densities were recorded in Nov 2012 and Jun 2013 (2304 and 2465 ind. ha -1 respectively). Successively lower densities were then recorded reaching densities approximately equal to those recorded in Jun 2012 (~1300 ind. ha - 1). Size distributions were small in Nov 2011 and Nov 2012, before increasing again to large sizes in Oct 2014. All size distributions had high-BL outliers and no small outliers. Scotoplanes globosa BL ranged from 1.3 to 19.3 cm.

3.2.2. Immigration and juvenile recruitment Numerous sampling periods in our study were associated with increases in both density and BL of holothurians. We tentatively identified these cases as representing some form of immigration prior to Nov 2011 ( Elpidia sp. A), Jun 2012 ( P. gracilis ), Nov 2012 ( Elpidia sp. A, Peniagone sp. 1), Jun 2013 (S. globosa , P. gracilis ), and Oct 2014 ( P. confundens , P. vitrea , Peniagone sp. A, and Peniagone sp. 1). The most notable examples of this pattern occurred in Elpidia sp. A, which significantly increased in BL while more than doubling in density twice during our study (in the 5 –6 months prior to Nov 2011 and Nov 2012), and P. gracilis for which Jun 2013 density was nearly three times that in Nov 2012, and BL was significantly higher. Two surveys in which we recorded high Peniagone sp. 1 density following from previously undetectable levels appeared to be aggregations of gravid individuals, possibly for spawning (Fig. 3) . We identified numerous periods in which a recent juvenile recruitment event appeared to occur based on a concurrent increase in density and reduction of median BL. Recruitment events might have occurred prior to Feb 2009 ( A. abyssorum ), Nov 2011 ( S. globosa , P. papillata , Peniagone sp. A), June 2012 ( A. abyssorum ), Nov 2012 ( A. abyssorum , P. confundens , S. globosa , P. vitrea , Peniagone sp. A), Jun 2013 ( P. papillata ) and Oct 2014 (Synallactidae sp. 1).

3.3. Body size and density

For the 15 holothurian species that were encountered in more than two sampling periods, maximum density was significantly inversely correlated with minimum BL (Spearman’s ρ = -0.9159, two-sided p < 0.0001; Fig. 4A —gray dots), mean BL (Spearman’s ρ = -0.7979, two-sided p = 0.0004; Fig. 4A —open circles), and median BL (Spearman’s ρ = -0.6676, two- sided p = 0.0065; Fig. 4A —black triangles). All 13

holothurians in our study that attained densities of 1000 or greater individuals ha -1 had a minimum body size of 1.5 cm or smaller.

Patterns in density across all sampling periods clustered by species with similar minimum BL records (Fig. 4B). Those species with minimum BL records between 0.5 –1.5 cm formed a significant cluster (Fig. 4C —red branching), and attained the highest maximum densities (Fig 4A). This cluster was comprised entirely of elpidiids. Those species with minimum BL records ranging from 1.8 –3.2 cm (Fig. 4B —blue branching) also formed a significant cluster, and attained moderate maximum densities (Fig 4A). Two elpidiids ( P. papillata and Peniagone sp. 2) were present in this cluster. Psychropotes longicauda and P. mollis had the largest minimum BL records (3.8 cm and 6.9 cm respectively), and were not a part of any significant cluster (Fig. 4A,B).

3.4. Holothurian community change

Diversity (Shannon H') of holothurians was not significantly different from 2006 –2009 versus 2011 – 2014 (average 1.56 ± 0.24 S.D.; Wilcoxon-Mann-Whitney U = 18.00, two-sided p = 0.44). Evenness was significantly higher from 2006 –2009 versus 2011 –2014 (Wilcoxon-Mann-Whitney U = 28.00, two-sided p = 0.0082; 2006 –2009 Pielou's J' = 0.84 ± 0.07 S.D; 2011 –2014 Pielou's J' avg. = 0.58 ± 0.10 S.D). The relative density patterns of holothurians during the 2006 –2009 period differed significantly from those documented from 2011 –2014 based on clustering at the 50% level (Fig. 4C).

4. Discussion

4.1. Holothurian densities from 2006 –2009 versus 2011 –2014

Our results suggest that a recent shift in the megafauna community at Sta. M (Kuhnz et al. , 2014) resulted in part from responses across multiple holothurian species. Between 2006 and 2009, sponges were the numerically dominant megafauna group (Kuhnz et al. , 2014). At that time, all holothurian species examined here were found in relatively low densities. Following 2009, holothurians as a group increased in density and gained numerical dominance, while sponges declined in density and proportional abundance (Kuhnz et al. , 2014). This transition involved significant increases in the densities of ten out of 19 holothurian species encountered from 2011 –2014, including all eight species within the family Elpidiidae. Ten species increased in density between Jun and Nov 2012, a period of exceptional availability of freshly-settled phytodetritus (Smith et al., 2014). We did not find a suite of holothurian species that thrived in 2006 –2009 but declined in 2011 –2014. Only one holothurian species ( P. mollis ) declined in density during this transition, and was less dense from 2011 –2014 compared to 2006 –2009. 14

These pervasive increases in holothurian density over a period lasting at least four years are comparable to what has been called the “ Amperima event” at the Porcupine Abyssal Plain (PAP), which lasted approximately three years. This event involved increases in densities of A. rosea , Ellipinion molle (Théel, 1879), P. longicauda , Oneirophanta mutabilis Théel, 1879, Pseudostichopus aemulatus Solis-Marin & Billett in Solis-Marin et al., 2004, and Molpadiodemas villosus (Théel, 1886), and decreases in Peniagone diaphana (Théel, 1882) (Billett et al. , 2001; Billett et al. , 2010). The abyssal holothurian densities we observed were comparable to those documented in other studies. Except for higher densities of A. abyssorum (1317 ind. ha -1) found at Sta. M in Jun 2002, our density estimates were similar to those documented previously at this site using towed camera sleds (Ruhl, 2007), and slightly higher than those found during trawl surveys of the same species or congeners in the northeast Atlantic at the PAP (Billett et al. , 2001; Billett et al. , 2010). All but two elpidiids in our study ( P. papillata and Peniagone sp. 2) exceeded densities of 1000 ind. ha -1 at some point, with the highest values calculated for Peniagone sp. A (11,627 ind. ha -1) and Elpidia sp. A (10,524 ind. ha -1). Very high densities are commonly attained by members of this family at sites throughout the globe, with some species far surpassing densities observed at Sta. M. The small elpidiid Amperima rosea (R. Perrier, 1896) in the abyssal Atlantic rose from undetectable levels in 1994 to over 6000 ind. ha -1 in 1997 as calculated using time-lapse photography (Bett et al. , 2001). Based on video and photographic records respectively, Kolga hyalina Danielssen & Koren, 1879 [following identification in Rogacheva (2012)] frequently reaches densities equivalent to 5,000 –10,000 ind. ha -1 in the Norwegian-Greenland Basin (identified as Irpa abyssicola Danielssen & Koren, 1879 in Gebruk et al. , 2003), and Peniagone japonica Ohshima, 1915 has reached estimated densities of 79,070 ind. ha -1 in Japanese waters (Ohta, 1983). Rhipidothuria racovitzai Hérouard, 1901 (then treated as Achlyonice violaecuspidata Gutt, 1990) and Elpidia glacialis Théel, 1876 in the Weddell Sea have been photographed at densities equivalent to 260,000 and 320,000 ind. ha -1 respectively (Gutt and Piepenburg, 1991). Perhaps attaining the highest known deep-sea holothurian densities, Kolga nana (Théel, 1879) has reached average densities equivalent to 500,000 ind. ha -1 as calculated from epibenthic sledge and photographic surveys in the Porcupine Seabight [(treated as Kolga hyalina in Billett and Hansen, 1982, taxonmy reassessed in Rogacheva (2012)]. Density estimates based on photogrammetry, epibenthic sledge, and trawl samples will differ (Billett, 1991) but gross differences can still be informative. As is common among echinoderms (Uthicke et al. , 2009) including abyssal holothurians (Billett et al. , 2001; Billett et al. , 2010), boom and bust cycles were typical among holothurians at Sta. M, especially among the elpidiids. This pattern was most-clearly demonstrated by Elpidia sp. A, for which density was higher by an order of magnitude in Nov 2012 compared to May 2011, and then was lower by roughly an order of magnitude in each subsequent survey through Oct 2014, when it was detected at very 15

low density. This species demonstrated a similar pattern previously at Sta. M (Ruhl and Smith, 2004). We do not know whether “bust” per iods represented mortality or emigration. Both of these processes remain poorly understood but are likely to be important components of holothurian population dynamics at Sta. M. In both time-lapse camera images [methods described in (Smith and Druffel, 1998)] and ROV video recorded at the site we have documented what might be dead holothurians on the sea floor. However individuals showing obvious signs of decay have been encountered rarely. We have seen many Peniagone sp. A lying on their side or rolling along the bottom, but we did not annotate these individuals as dead in ROV footage. They did not look compromised otherwise. Those individuals might have been knocked over by the bow wave of the ROV, or performing a behavior we have observed in time-lapse images but cannot explain: individuals of Peniagone sp. A have been seen lying on their side for several hours and appearing dead, but then resuming a prone position and crawling or swimming away.

4.2. Indicators of immigration and juvenile recruitment

Juvenile recruitments of multiple species appear to have occurred during three periods in our study (prior to Nov 2011, Nov 2012 and Jun 2013). All of these periods occurred between 2011 and 2014. Small sample sizes might have contributed to our failure to detect apparent recruitment events (or a lack thereof) between 2006 and 2009. Abyssocucumis abyssorum , P. papillata , Peniagone sp. A, and S. globosa appear to have demonstrated more than one recruitment event each from 2011 –2014, suggestive of opportunistic reproduction or settlement by these species when food was plentiful. Evidence for reproductive recruitment of multiple holothurian species has been documented previously at the PAP (Billett et al. , 2001; Billett et al. , 2010), and Sta. M (Jun 1990; Ruhl 2007). The “ Amperima event” involved recruitments of both A. rosea and P. longicauda (Billett et al. , 2010), perhaps during a shift in food composition at the PAP (Wigham et al. , 2003a). In Jun 1990 at Sta. M, a period of moderate food supply, Elpidia sp. A (then identified as E. minutissima ), P. diaphana , P. vitrea , and A. abyssorum exhibited both lower median BL and higher densities compared to the prior sampling period (Ruhl, 2007). Based on examples for which density and BL distribution were both higher in consecutive sampling periods, we documented possible immigration of six species of holothurian ( Elpidia sp. A, P. confundens , P. gracilis , Peniagone sp. A, Peniagone sp. 1, and P. vitrea ), an assemblage that is skewed toward species that attained high maximum densities (Table 2), exhibited similar density patterns (Fig. 4A), and had small minimum BL records (Fig. 4A; Supplementary Table 1). For all of these species, maximum densities coincided with possible immigration events (Fig. 2, Fig. 4C). Furthermore adult Peniagone gracilis , Peniagone sp. A, Peniagone sp. 1, P. vitrea , and P. confundens are known to swim as adults (Table 2; Miller and Pawson, 1990). We do not know the distances from which these holothurians 16

might have emigrated, or the degree to which benthopelagic species might have swum to new areas. Sta. M site is an open system without obvious boundaries to the surrounding abyssal benthos.

The idea that deep-sea holothurians might migrate and/or form spawning aggregations is not new (Billett, 1991; Ruhl, 2007). Numerous echinoderms aggregate (Billett and Hansen, 1982; Gutt and Piepenburg, 1991; Young et al. , 1993) for purposes including reproduction (Tyler et al. , 1993). Some cold-water holothurians undergo vertical depth migrations for spawning (Hamel and Mercier, 2008). Deep-sea holothurians can exhibit patchy or aggregated distributions in relation to food at Sta. M (Lauerman et al. , 1996). Distant chemical cues have been shown to assist cold-water echinoderms in locating food [e.g. the sea star Odontaster validus Koehler, 1906 (Kim et al. , 2007)], and inducing spawning (Soong et al. , 2005) at the local scale. However the bathyal echinoid Stylocidaris lineata does not appear to locate food from a distance (Young et al. , 1993). To our knowledge the role of distance chemoreception in influencing deep-sea holothurian migration and/or aggregation has been hypothesized (Ohta, 1985), but not explored. The ability of some holothurians to swim has been suggested as a factor that might facilitate migrations (Billett, 1991), and the tendency of numerous species to move or orient consistently according to currents (Ohta, 1983; Gutt and Piepenburg, 1991; Kaufmann and Smith, 1997; Gebruk et al. , 2003; Gallo et al. , 2015) provides a possible mechanism for navigation. The empirical study of large-scale movements by adult holothurians represents a significant opportunity for further understanding of true holothurian biomass distribution, and the scale of their influence on abyssal ecosystems.

Multi-species recruitment and immigration events were documented during 2011 –2014, but only once during 2006 –2009 (one recruitment of A. abyssorum prior to Feb 2009). Throughout this period, Nov 2012 appears to have been the most active time for recruitment and immigration of holothurians to Sta. M, with increases in 10 out of 19 species. This survey period followed shortly after continuous, high percent-coverage depositions of surface-derived salp detritus and phytodetritus were observed on the sea floor, lasting from Mar –Aug 2012 (Smith et al. , 2014). These high-flux deposition events fueled the most significant period of food over-supply documented at Sta. M. since records began in 1989 (Smith et al. , 2013). We can only speculate that these ten species might have responded to the same environmental cue to increase their local densities: a high magnitude deposition of high quality food to the sea floor. In conjunction with this event, density of Peniagone sp. A increased rapidly, lagging salp detritus percent cover by 14 weeks (Smith et al. , 2014). This rapid increase in density was accompanied by a significant reduction of median BL between Jun and Nov 2012. Based on these results we raise the possibility that larval settlement of Peniagone sp. A might have occurred in response to these deposition events. If 17

Peniagone sp. A recruitment stemmed from local reproduction, then an opportunistic spawning response by Peniagone sp. A took place on a very short timescale.

We recognize several sources of error that might have impacted our assessments of recruitment and immigration. We assumed only positive growth when interpreting patterns in BL. However body length of individual holothurians can change significantly through uptake or excretion of water (Hamel et al. , 1993), and negative growth can occur during periods of non-feeding (So et al. , 2010; Hannah et al. , 2012). Assimilation efficiencies can be negative in the absence of phytodetritus (Ginger et al. , 2001). If negative growth occurred at Sta. M during our study then we might have falsely interpreted declines in median BL as resulting from recruitment events. Because spatial patchiness can be common among deep- sea holothurians, and has occurred at Sta. M ( Lauerman et al., 1996), demographics surveys can be sensitive to deviations of survey location (Billett and Hansen, 1982). Calculating densities for species that aggregate can be especially problematic because individuals concentrate from a larger area of unknown spatial extent [e.g. (Ohta, 1985; Billett and Hansen, 1982)]. Elsewhere megafauna spatial variation has been attributed to influences of bottom topography on food availability (Rowe, 1971; Billett and Hansen, 1982) and flow dynamics, which might alter particulate supply and sediment characteristics (Durden et al. , 2015a). Body size (Billett and Hansen, 1982; Bisol et al. , 1984), and reproductive characteristics (Galley et al. , 2008; Baillon et al. , 2011) can also vary spatially. These aspects are not expected to have a significant impact on our results and conclusions. Station M lacks substantial relief (Kaufmann and Smith, 1997), and of the holothurians we documented, only Peniagone sp. 1 in Nov. 2012 was strongly aggregated (Fig. 3). Spatial heterogeneity in density and dispersion of individual megafauna taxa can be significant over a distance of tens of kilometers at Sta. M, although near-synoptic megafauna density measures from nearby sites agreed by an average of 70% (Lauerman et al. , 1996). Our study was based on benthic transects confined to a relatively small area compared to earlier towed-camera sled studies at the site (Kuhnz et al. , 2014). Finally, we assumed any spatial variation in BL and densities to be overshadowed by temporal variation in these aspects as found during a 16 yr. time series study of BL and densities at Sta. M (Ruhl, 2007). The interannual fluctuations observed here far exceeded the spatial variation reported previously (Lauerman et al. , 1996).

4.3. Body size and density

Small body size, continuous gamete production, broadcast spawning, and a brief lecithotrophic larval stage are considered important factors allowing some deep-sea holothurians to recruit opportunistically high numbers of young to the sea floor during unpredictable favorable conditions (Billett and Hansen, 1982; Tyler et al. , 1985a; Billett, 1991; Gebruk et al ., 2003; Hudson et al. , 2003; 18

Wigham et al. , 2003b; Ruhl, 2007; Billett et al. , 2010; Rogacheva et al. , 2012b). Our results add to these ideas by showing significant inverse correlations between body size measures and maximum densities attained.

4.4. Holothurian community change

At the abyssal monitoring sites Sta. M and the PAP, abrupt and long-lasting transitions in holothurian communities have been traced to changes in surface processes that influence the quantity, quality, timing, and distribution of phytodetritus settled on the sea floor (Gage and Tyler, 1991; Billett et al. , 2001; Wigham et al. , 2003a; Ruhl and Smith Jr, 2004; Smith et al. , 2006; Ruhl, 2007; Ruhl et al. , 2008; Billett et al. , 2010). These changes have led to community-wide alterations in reproductive biology, species composition, dominance, abundance, carbon burial (Ruhl, 2007; Ruhl et al. , 2008; FitzGeorge- Balfour et al. , 2010) and possibly animal distribution and movements (Lauerman et al. , 1996; Kaufmann and Smith, 1997). Our study lends further support to these ideas. Recent anomalous surface processes along the U.S. west coast (e.g. Chavez et al. , 2011; Kahru et al. , 2012; Kintisch, 2015) have led to increases in food flux events at Sta. M (Smith et al. , 2013; Smith et al. , 2014), and corresponded with significant and persistent changes in holothurian assemblages. Holothurian communities during the 2006 – 2009 period were significantly different from those documented between 2011 and 2014 (Fig. 4B). These periods corresponded with periods of moderate and exceptionally high food supply respectively (Smith et al. , 2013; Smith et al. , 2014). While species diversity of holothurians remained unchanged during our study, species evenness was significantly lower in 2011 –2014, as has been found previously during periods of elevated food supply at Sta. M (Ruhl, 2008). These patterns largely agree with the theoretical framework outlined by (Ruhl et al. , 2013) predicting relationships between total density, respiration rate, and species evenness.

We cannot evaluate our results separately from a phylogenetic context. The ecological patterns in our study were dominated by significant increases in densities of elpidiid holothurians when food availability was very high, following from very low or undetectable densities of these species during a period of moderate supply. While two non-elpidiid species showed significant increases in density, other families did not similarly exhibit unanimously higher densities in 2011-2014 compared to 2006-2009. Traits conferring ecological advantages to elpidiids during periods of high food supply, and conversely those that might limit maximum densities of other species, are likely to be phylogenetically constrained (Eckelbarger and Watling, 1995). The ability of elpidiids to select for (Iken et al. , 2001) and monopolize consumption of fresh phytodetritus (Wigham et al. , 2003a; Wigham et al. , 2003b) through increased feeding activity during high-food flux events (e.g. Kaufmann and Smith, 1997; Bett et al. , 2001) leads to 19

clear ecological advantages (Billett and Hansen, 1982; Billett, 1991; Billett et al. , 2010). Likewise their characteristic opportunistic reproductive strategies (Galley et al. , 2008) involving small egg size (Hansen, 1975; Billett and Hansen, 1982; Tyler and Billett, 1988; Wigham et al. , 2003b), potentially short-duration larval stages (Wigham et al ., 2003b), small body size (Gebruk et al. , 2003), and potentially small size at recruitment (this study) might allow for accelerated generation times. Some holothurians outside of Elpidiidae might be physiologically limited in their ability to produce large numbers of eggs, as has been shown in O. mutabilis (Ramirez-Llodra et al. , 2005), which broods its young, and psychropotids, which produce exceptionally large eggs (Hansen, 1975; Tyler et al. , 1985b). Rapid locomotion and feeding behavior have been predicted to confer trophic advantages to O. mutabilis exploiting detritus on the sea floor (Roberts et al ., 2003). These advantages did not translate to higher population densities of this species complex when fresh phytodetritus was available in great quantities at Sta. M. Any competitive advantage of elpidiids conferred by the characteristics above might be short-lived, or strongly dependent on the availability of fresh surface-derived nutrients. Elpidiids were found in very low or undetectable numbers when fresh phytodetritus was limiting, and they exhibited significant boom and bust cycles when it was abundant. From an evolutionary standpoint, if Elpidiidae forms a monophyletic group then many traits considered amenable to high holothurian density on the abyssal sea floor during boom times might have evolved once, in the most recent common ancestor of elpidiids. These traits would therefore represent a sample size of one in comparative studies (Garland et al. , 1992). A detailed understanding of deep-sea holothurian phylogenetic relationships is required for studies using independent contrasts (Garland et al. , 1992) to yield a more robust understanding of deep-sea holothurian responses to food supply.

4.5. Conclusions

We studied change in densities and BL demographics of holothurians from 2006 –2009 and 2011 – 2014, periods that took place before and after a recent shift in the megabenthic community at the abyssal time-series monitoring site Sta. M (Kuhnz et al. , 2014). This latter period encompassed exceptionally high-magnitude pulses of food supply to the sea floor (Smith et al. , 2013; Smith et al. , 2014). While holothurian species diversity did not change during our study, these animals exhibited lower densities and higher species evenness during 2006 –2009 than 2011 –2014. Patterns indicative of juvenile recruitment and immigration by multiple species were exhibited multiple times when food was abundant. This pattern was dominated by species within the elasipodid family Elpidiidae, which were present in very low densities during 2006 –2009 and very high densities during 2011 –2014. Species in this group might have exhibited opportunistic responses to unpredictable food supply through heightened recruitment and 20

immigration to the area, perhaps facilitated by their relatively small body size and small size at juvenile recruitment.

5. Acknowledgements

This research was funded by the David and Lucile Packard Foundation. L.L. was supported by the Monterey Bay Aquarium Research Institute Summer Internship Program. We thank the crew of the RV Western Flyer , and the pilots of ROVs Tiburon and Doc Ricketts for their continued support. This paper was improved by the constructive comments of Henry Ruhl and three anonymous reviewers.

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Figure legends:

Figure 1. Holothurian densities at Station M based on remotely operated vehicle transects at Sta. M between 2006 –2014. Dashed red line highlights density of 1000 ind. ha -1. Species represented by warm colors exceeded densities of 1000 ind. ha -1 at some point during the study.

Figure 2. Density and body length (BL) distributions of holothurians encountered during remotely operated vehicle transects at Sta. M between 2006 –2014 (density in red, notched box plots of BL in black). Box plots are shown for samples sizes of 10 or greater showing sample mean (+), whiskers (1.5 x interquartile range), and outliers (open circles). Letters by box plots identify groups significantly different from each other based on non-overlap of box plot notches. Letters signifying size distributions are in alphabetical order with group A representing the group with the largest individuals and E the smallest of each species. Small individuals of A. abyssorum were undersampled because they were frequently found on top of other objects and could not be sized. Small individual A. abyssorum were especially undersampled in Feb 2009, Nov 2011, Nov 2012 and Apr 2014 when 85%, 25%, 30% and 32% of sampled individuals could not be sized (see text).

Figure 3. Peniagone sp. 1. (A) Close up and (B) wider view showing high densities of what appear to be gravid individuals in Nov 2012. Targeted collections verified the presence of ripe eggs and sperm. Red lasers in panel B are 29 cm apart.

Figure 4. Density patterns of holothurians encountered during remotely operated vehicle transects at Sta. M, from 2006 –2014. A) Scatter plot of maximum density recorded versus minimum (gray circles), mean (open circles) and median (black triangles) body length (BL). Note common log scale of axes. Colored lines were added on the y-axis for illustrative purposes to identify maximum density ranges of species that clustered by density patterns according to the SIMPER test. Line color corresponds with dendrogram branch color in panel C. B) Clustering of holothurian species according to similarities in their density patterns across remotely operated vehicle transects at Sta. M between 2006 –2014. Red and blue dashed lines identify clusters that are significantly similar to the degree indicated on the y-axis, according to the SIMPER test. An asterisk denotes species known (or suspected in parentheses) to swim in their adult form based on in situ observations at Sta. M or a benthopelagic designation by Miller and Pawson (1990). C) Multidimensional scaling plot comparing holothurian density patterns by sampling period (2006–2009 black squares; 2011 –2014 green squares; each square represents a separate ROV survey). Ellipsoids group data points that cluster according to the SIMPER test (40% similarity- gray lines, 50% similarity- black lines).

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Table 1. Survey periods and seafloor transect coverage. Dive numbers starting with “T” were conducted with ROV Tiburon , and those started with “D” were conducted with ROV Doc Ricketts .

2 Dive Period Area (m ) T1067 Dec-06 1120 T1079, T1080 Feb-07 220 T1141, T1143 Sep-07 420 D8 Feb-09 1560 D230-232 May-11 1830 D321 Nov-11 1004 D403 Jun-12 400 D442 Nov-12 1377 D486 Jun-13 1278 D581 Apr-14 1414 D671-674 Oct-14 1340

Table 2. Densities of holothurians (ind. ha -1) recorded at Station M between Dec 2006 –Oct 2014 as calculated from ROV transects of sea floor. Those species in bold were encountered in significantly different densities in 2011 –2014 compared to 2006 –2009 (- = species with greater density in 2006 –2009 compared to 2011 –2014; + = species with greater density in 2011 –2014 compared to 2006 –2009; † = those known to bury; * = those known to be capable of swimming in their adult form based on in situ observations in California, designation as benthopelagic in the adult form (Miller and Pawson, 1990), or suspected (in parentheses) based on the presence of a velum (following Rogacheva and Gebruk, 2013).

Order Family Dec Feb Feb Apr - - Sept - May - 200 200 - 200 - Nov- Jun- Nov- Jun- 201 Oct- Taxon 6 7 2007 9 2011 2011 2012 2012 2013 4 2014 Dendrochiroti Cucumariidae †Abyssocucumi da s abyssorum 125 227 95 269 84 80 175 177 180 42 104 Aspidochiroti Synallactidae +*Paelopatides da confundens 0 91 24 6 11 80 50 112 86 106 134 - Pseudostichop us mollis 36 0 24 71 11 0 0 0 0 0 15 Synallactidae sp. 1 45 182 95 38 55 10 25 67 94 71 172 Elasipodida Deimatidae Oneirophanta mutabilis complex 27 45 0 51 16 10 50 0 63 7 30 Pelagothuriid *Enypniastes ae sp. 9 0 0 0 0 0 0 0 0 0 0 Psychopodida Psychropotes e depressa 0 0 0 0 0 10 0 0 0 0 0 +Psychropotes longicauda 0 0 0 0 0 0 25 37 23 21 7 Elpidiidae 1052 +Elpidia sp. A 0 409 0 109 1295 3436 4725 4 4264 141 22 +*Peniagone gracilis 0 0 0 0 907 777 1150 337 900 375 306 +*Peniagone papillata 36 0 24 13 224 309 150 105 141 57 22 +*Peniagone vitrea 45 0 0 410 732 966 800 1683 1088 389 2545 *Peniagone 525 1162 sp. A 18 0 0 19 3628 4213 4200 5079 5321 5 7 28

+*Peniagone sp. 1 0 0 0 0 0 0 0 2917 1534 7 1142 +(*)Peniagone sp. 2 0 0 0 19 11 159 50 202 55 57 22 +Scotoplanes 133 globosa 0 0 0 6 514 876 1300 2304 2465 7 1246 UNK UNK Holothuroidea sp. 4 18 0 24 90 33 70 25 22 55 7 22 Holothuroidea sp. 5 0 0 0 27 13 0 0 0 0 0 0 Holothuroidea sp. 6 0 0 0 6 0 0 0 0 0 0 0 113 1099 1272 2356 1626 787 1741 TOTAL 359 954 286 4 7534 6 5 6 9 2 6

Highlights

 We studied change in holothurian demographics at the time-series site Station M.  2006-2009 had lower densities and higher species evenness than 2011-2014, high-food years.  Maximum densities were significantly inversely correlated with minimum body length.  High-food years exhibited periods indicative of recruitment and immigration by multiple species.  Responses to high-food conditions took place primarily in the group Elasipodida: Elpidiidae.

Supplementary Figure 1. Scatter plot of median, minimum, and mean body length (BL) of holothurians at Sta. M between 2006 and 2014

A Non ‐elasipodid Paelopatides confundens holothurians Pseudostichopus mollis Abyssocucumis abyssorum 100,000 Synallactidae sp. 1 Holothuroidea sp. 4 10,000

1,000 ) -1 100

10

0 Density(ind. ha

2007 2008 2009 2010 2011 2012 2013 2014 201 5

B Elasipodida: Psychropotes longicauda Non-Elpidiidae Oneirophanta mutabili s complex 100,000

10,000

1,000 ) -1 100

10

0 Density(ind. ha

2007 2008 2009 2010 2011 2012 2013 2014 201 5

Elpidia sp. A C Elasipodida: Peniagone gracilis Elpidiidae Peniagone papillata Peniagone vitrea Peniagone sp. A Peniagone sp. 1 100,000 Peniagone sp. 2 Scotoplanes globosa

10,000

1,000 ) -1 100

10

0 Density (ind. ha (ind. Density

2007 2008 2009 2010 2011 2012 2013 2014 201 5 Sampling period (year) length (cm) length (cm) length (cm) length (cm) 10 15 10 0 10 15 20 25 20 length (cm) 10 15 5 5 0 0 5 10 15 20 25 30 0 2 4 6 8 0 5 B Elpidia Elpidiidae: Abyssocucumis abyssorum Cucumaridae: Scotoplanes globosa Elpidiidae: Synallactidae Synallactidae: Paelopatides confundens Synallactidae: 07 20 09 21 01 21 03 21 2015 2014 2013 2012 2011 2010 2009 2008 2007 + C + + + + + + + + sp. A sp. 1 C + + + + + 1 Sampling period(year) C A B A + + + + + B B D + + + + + B B + + + + + A B B C + + + + + A A A B B + + + + + A B B B + + + + + A B A B + + + + + 100 120 140 160 100 150 200 250 300 50

0 10000 12000 100 150 200 2000 4000 6000 8000 50 0 1000 1500 2000 2500 20 40 60 80 0 0 500 0

-1 Ind. ha -1 Ind. ha -1 Ind. ha -1 Ind. ha Ind. ha -1 length (cm) length (cm) length (cm) 10 12 length (cm)

10 20 30 40 length (cm) 0 2 4 6 8 10 0 10 15 20 25 30 10 12 0 2 4 6 8 5 0 2 4 6 8 0 Peniagone vitrea Elpidiidae: 07 20 09 21 01 21 03 21 2015 2014 2013 2012 2011 2010 2009 2008 2007 + Peniagone Peniagone Elpidiidae: Peniagone papillata Elpidiidae: Peniagone Peniagone Elpidiidae: Peniagone gracilis Elpidiidae: + + + + sp. 1 sp. A D + + Sampling period(year) B A A A + + + + A D B B + + + + B C A + + + + B C B C D + + + + + C B B C B + + + + + C D E + + + + + A B A A + + + + + 10000 12000 1000 2000 3000 1000 1500 2000 2500 3000 3500 2000 4000 6000 8000 0 0 500 1000 1200 100 150 200 250 300 350 200 400 600 800 0 0 50 0

Ind. ha -1 Ind. ha -1 Ind. ha -1 Ind. ha -1 Ind. ha -1 A B A C Min BL 1e+5 Mean BL Median BL

1e+4 ) -1

1e+3

1e+2 Maximumdensity (ind. ha 1e+1 Transform: Square root 2D Stress: 0.03 Similarity 2006-2009 10 100 40 0.1 1 Resemblance: S17 Bray Curtis similarity 2011-2014 body length (cm) 50

B 20 Holothurian density Transform: Square root Min BL (cm) All sampling periods Resemblance: S17 Bray Curtis Similarity 0.5 0.9 1 40 1.2 1.3 1.5 60 1.8

Similarity 2.3 2.5 2.6 80 2.8 3.2 3.8 100 6.9 swims *

sp. 1 sp. A sp. A sp. 2

Elpidia Peniagone Peniagone Peniagone vitrea ( ) Peniagone * Peniagone* gracilis * SynallactidaePeniagone sp. 1 papillata * * Holothuroidea sp. 4 * Scotoplanes globosa Pseudostichopus mollis Oneirophanta mutabilis Psychropotes longicauda complex Paelopatides confundens Abyssocucumis abyssorum * 20 Density patterns cluster by minimim body length (BL) in abyssal holothurians Min BL (cm) 0.5 0.9 1 1.2 40 1.3 1.5 1.8 2.3 60 2.5

Similarity 2.6 2.8 3.2 3.8 80 6.9

100