TROPHIC STRUCTURE OF MIDWATER FISHES OVER COLD SEEPS IN THE NORTH CENTRAL GULF OF MEXICO. Jennifer P. McClain-Counts

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1 TROPHIC STRUCTURE OF MIDWATER FISHES OVER COLD SEEPS IN THE NORTH CENTRAL GULF OF MEXICO Jennifer P. McClain-Counts A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Master of Science Center for Marine Science University of North Carolina Wilmington 2010 Approved by Advisory Committee Steve W. Ross Chair Lawrence B. Cahoon Joan W. Willey Accepted by Dean, Graduate School

2 TABLE OF CONTENTS ABSTRACT... iv ACKNOWLEDGMENTS... vi DEDICATION... vii LIST OF TABLES... viii LIST OF FIGURES... xi INTRODUCTION...1 METHODS...4 Study Area...4 Sample Collection...5 Dietary Analyses...6 Gut Content Analyses...6 Stable Isotope Analyses...7 IsoSource Mixing Model...10 Trophic Position Analyses...10 Statistical Analyses...11 RESULTS...13 Catch Data...13 Gut Content Analyses...13 Diet composition...13 Factors influencing diet composition...17 Stable Isotope Analyses...19 Trophic Position Calculations...23 ii

3 DISCUSSION...24 Diet composition...24 Spatial and Temporal influences on diet...30 Additional insight with SIA...32 Site differences...32 Diet variations...33 Methodology...34 Interesting Note...35 CONCLUSIONS...36 LITERATURE CITED...37 iii

4 ABSTRACT Midwater fishes are an important component of pelagic food webs and provide insight into energy utilization and movement through the water column. In this study, the diets of midwater fishes collected over cold seep habitats were examined to determine general feeding patterns and whether size, depth, time of day or location affected diet composition within fish species. The base of the midwater food web was also examined to determine whether chemosynthetic energy in benthic cold seeps was incorporated into the midwater fish community. Discrete depth Tucker trawling was conducted in August 2007 over three cold seep habitats (> 1000 m) in the northcentral Gulf of Mexico. Surface sampling was also conducted to provide a prey base (zooplankton and POM) for stable isotope analyses (SIA). Gut content analysis (GCA) and SIA (δ 13 C and δ 15 N) in conjunction with IsoSource software were utilized for diet reconstruction and to determine trophic positions. SIA also aided efforts to determine chemosynthetic influences on the midwater food web. GCA was performed on 31 species in the five most abundant families (Gonostomatidae, Myctophidae, Phosichthyidae, Sternoptychidae and Stomiidae), with midwater fishes classified into one of three guilds: piscivore, large crustacean consumer, or zooplanktivore. SIA was performed on 6 fish families (Gonostomatidae, Myctophidae, Phosichthyidae, Sternoptychidae, Stomiidae, and Melamphaidae), 13 invertebrate categories, and 3 primary producers (POM, Sargassum spp. and detritus), and classified all fishes as zooplanktivores. Using IsoSource, more precise contributions of individual prey taxon were documented, which did not always support results from GCA. Size, depth, time of day and location did not affect diet composition within a species; however migration trends suggested competition may be reduced by feeding over a range of depths and over a 24 hour period. Significant differences in trophic position calculations between GCA and SIA highlighted the iv

5 importance of using multiple techniques to describe trophic structure, as each method characterized the diets differently. v

6 ACKNOWLEDGMENTS This project was largely funded by the Department of the Interior U.S. Geological Survey under Cooperative Agreement No. 05HQAG0009, sub agreement 05099HS004. I thank the crew of the R/V Cape Hatteras and all scientific personal for assisting with fishing operations and sample processing. S. Artabane, A. Quattrini, and A. Roa-Varon assisted with fish identifications and C. Ames assisted with invertebrate identifications. Guidance and support during stable isotope analyses were provided by Drs. A. Demopoulos and C. Tobias, and K. Duernberger. I would also like to thank S. Artabane, T. Casazza and A. Roa-Varón for their assistance in dissecting and processing fish stomachs. Special thanks to my committee, Drs. S. Ross, L. Cahoon, and J. Willey, for their guidance and support during the duration of this project. I would additionally like to thank my advisor, Dr. S. Ross, for setting me up with this project and Dr. L. Cahoon for his assistance with statistics. Finally, thanks to S. Ross, T. Casazza, A. Demopoulos, A. Quattrini, L. Truxal and M. Carlson for their suggestions and edits provided throughout the writing process of this thesis. vi

7 DEDICATION I would like to dedicate this thesis to my parents, who encouraged my early passion in marine science and gave me the confidence to follow my dreams and overcome any obstacles. Your constant love and support was unwavering and because of that, I can present this Masters project. vii

8 LIST OF TABLES Table Page 1. Surface and midwater stations sampled over three cold seep sites (AT340, GC852, and AC601) (see Fig.1) in the Gulf of Mexico (9-25 August 2007) The total number of all midwater fishes, invertebrates and autotrophs examined in dietary analyses from the North-central Gulf of Mexico Results of ANOSIM comparing effects of size, time of day, depth and location on the general prey categories consumed for each fish species Percent volume and frequency of prey items consumed by Chauliodus sloani collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Gonostoma elongatum collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Stomiidae collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Cyclothone alba collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Cyclothone braueri collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Cyclothone pseudopallida collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Hygophum benoiti collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day...68 viii

9 11. Percent volume and frequency of prey items consumed by Valenciennellus tripunctulatus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Diaphus mollis collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Cyclothone pallida collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Vinciguerria poweria collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Myctophum affine collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Argyropelecus aculeatus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Argyropelecus hemigymnus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Pollichthys mauli collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Benthosema suborbitale collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Lampanyctus alatus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Lepidophanes guentheri collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day...88 ix

10 22. Percent volume and frequency of prey items consumed by Notolychnus valdiviae collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Percent volume and frequency of prey items consumed by Ceratoscopelus warmingii collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day Mean (± 1 SE) δ 13 C and δ 15 N values for midwater fishes, invertebrates and carbon sources collected from each site (AC601, AT340, GC852) Percent of prey contributions for each midwater fish species using IsoSource Mean trophic position (TP), one standard deviation (SD), range (minimum maximum) and number of fish (n) for each midwater fish species collected in the North-central Gulf of Mexico, using data from stable isotope and gut content analyses x

11 LIST OF FIGURES Figure Page 1. Sampling areas in the North-central Gulf of Mexico for midwater fauna, 9-25 August The three cold seep sites (AT340, GC852, AC601) were located on the continental slope at depths > 1000 m. Each dot represents one station Multidimensional scaling (MDS) plot documenting the differences among the gut contents of midwater fishes. Data were based on the Bray-Curtis similarity matrix calculated from standardized, square root transformed, mean volumes of prey (12 general categories) Relationships among stomach fullness, mean depth of capture and time for midwater fishes. Data were compiled from all sites and excluded specimens lacking depth data Plot of the average δ 15 N values against the average δ 13 C values (± 1 standard error) for midwater fishes, invertebrates and primary producers collected in the Gulf of Mexico Relationship between δ 15 N and SL for all midwater fish species xi

12 INTRODUCTION Midwater fishes constitute an important component of the pelagic food web due to their high abundances, migratory behavior, and global distribution (Gjøsaeter and Kawaguchi 1980; Cornejo and Koppelmann 2006). Many of these unique fishes inhabit the mesopelagic zone (200 to 1000 m), and although they are consumed by a variety of marine fauna, such as benthic grenadiers (Laptikhovsky 2005), pelagic tuna (Potier et. al. 2007), and penguins (Adams et al. 2004), midwater fishes are also fierce predators. In the eastern Gulf of Mexico, midwater fishes consume 5-10% of the daily zooplankton production in the epipelagic zone (< 200 m) (Hopkins et al. 1996). The ability of midwater fishes to impact both surface and bottom communities results from diel vertical migrations (DVMs), a unique behavior exhibited by many midwater fish species. Species that undergo DVMs migrate from the mesopelagic zone to the epipelagic zone at night, primarily at sunset, and return to the mesopelagic zone at sunrise. Through DVMs, midwater fishes, particularly myctophids (Kinzer 1977; Hidaka et al. 2001; Cornejo and Koppelmann 2006), contribute significantly to the vertical transport of organic matter from the epipelagic zone to the mesopelagic zone (Ashjian et al. 2002; Brodeur and Yamamura 2005), thus impacting the trophic structure of the water column. By tracking these trophic relationships, a more thorough understanding of pelagic energy and material flow through the water column can be established. Previous dietary studies on midwater fishes (Hopkins and Baird 1985a,b; Lancraft et al. 1988; Hopkins et al. 1996; Butler et al. 2001; Pusch et al. 2004) utilized gut content analyses (GCA) to determine trophic relationships. Generally, midwater fishes were divided into three major feeding guilds: zooplanktivores, which consume planktonic organisms such as amphipods, copepods and euphausiids; micronektonivores, which consume fishes and cephalopods; and 1

13 generalists, which consume a variety of unrelated taxa (Gartner et al. 1997). Unfortunately, as GCA only represent short-term diet (< 24 hours) (Hadwen et al 2007), placement into these feeding guilds could vary and may be inaccurate. Guild placement can be affected by dietary shifts resulting from changes in prey abundance (Kawaguchi and Mauchline 1982), seasonality (Kawaguchi and Mauchline 1982) and ontogeny (Kawaguchi and Mauchline 1982; Young and Blaber 1986; Hopkins et al. 1996; Beamish et al. 1999; Williams et al. 2001; Butler et al. 2001). Additionally, accurate guild placement is not possible for specimens with empty stomachs, common in midwater fishes (Gartner et al. 1997). The trophic relationships relating midwater fishes to their carbon sources are also limited with GCA, which may not always allow the determination of which autotrophs contributed to a food web (Thomas and Cahoon 1993). Therefore, despite providing detailed dietary data, GCA only documents a portion of the trophic structure. Issues related to GCA (noted above) may be addressed using stable isotope analyses (SIA). Although SIA cannot provide detailed prey data (e.g., species level prey identifications), SIA provides general information on the cumulative feeding habits of an organism (Fry 2006). Trophic positions within a food web (Fry 1988; Van Dover 2000; Hobson 2002; Behringer and Butler 2006; Fry 2006; Paradis et al. 2008) can be estimated from SIA due to the isotopic ratio of nitrogen ( 15 N/ 14 N or δ 15 N), increasing an average of 3.4 per trophic level (Minagawa and Wada 1984; Post 2002). In contrast to nitrogen, the isotopic ratio of carbon ( 13 C/ 12 C or δ 13 C), has little fractionation between trophic levels, with an increase 1 per trophic level (Post 2002; Lajtha and Michener 1994; Minagawa and Wada 1984). Despite this low fractionation, carbon is useful for determining carbon sources as distinct ranges are documented for different autotrophs: -22 to -16 for marine phytoplankton (Post 2002; Fry 2006), -18 to -15 for Sargassum spp. 2

14 (Rooker et al. 2006), -16 to -5 for turtlegrass (Hemminga and Mateo 1994), and -75 to -28 for chemosynthetic material (Kennicutt et al. 1992). Unfortunately, despite the added benefit of combining SIA and GCA in dietary analyses, few studies (e.g. Vander Zanden et al. 1997; Hadwen et al. 2007; Drazen et al. 2008; Rybczynski et al. 2008) have done so. In the Gulf of Mexico (GOM), trophic structure may be affected by the complex bottom topography and hydrography. The dominant current, the Loop Current, flows from the Caribbean Sea through the Yucatan Channel, around the east-central portion of the GOM and flows out near southern Florida (Hyun and Hogan 2008; Sturges et al. 2005). The oscillation of this current often results in warm- and cold-core rings breaking off, which affect circulation (Schmitz et al. 2005), primary productivity and food web dynamics (Waite et al. 2007). Additionally, the Mississippi River flows into the northern portion of the basin providing large amounts of freshwater, sediments and nutrients, affecting both the water physics and chemistry and faunal communities (Baguley et al. 2006; Jarosz and Murray 2005). The presence of benthic features, like cold seeps or corals, can also affect trophic complexity, particularly as chemosynthetic communities can be associated with cold seeps. Higher abundances of non-seep, benthic fauna were occasionally observed in the vicinity of seeps (Levin 2005) and may consume chemosynthetic material (MacAvoy et al. 2002; 2008). However, whether midwater fishes are impacted by chemosynthetic energy pathways either in the water column above the seep areas or by interactions with the associated benthic communities has not been examined. Food web studies provide an effective means of tracking energy flow through an ecosystem. The purpose of this study was to examine the trophic structure of midwater fishes over cold seep areas (> 1000 m) in the north-central GOM. The presence of changing hydrography and prey resources at the sites may affect trophic structure. This study used both GCA and SIA to 3

15 thoroughly document the trophic relationships of midwater fishes. The objectives were to: 1) determine basic feeding patterns of the dominant midwater fish species collected, 2) document feeding changes, if any, that occurred among species due to differences in size, time of day, depth or location, 3) examine the relationship, if any, between feeding and DVM patterns in the midwater fishes, 4) document the differences in short term (GCA) and long term (SIA) feeding, and 5) examine the base of the midwater food web to determine whether the midwater community utilized chemosynthetic energy sources from cold seeps. METHODS Study Area Three cold seep sites in the GOM were selected for sampling based on data collected by TDI- Brooks International, Inc: Atwater Valley Block 340 (AT340), Green Canyon Block 852 (GC852), and Alaminos Canyon Block 601 (AC601). These three sites are located on the middle to lower continental slope in the north-central GOM, and each contained benthic chemosynthetic communities (Fig. 1). Detailed bottom topography was documented for each site from previous seismic profiles and surveys from a submersible and a remotely operated vehicle (Roberts et al. 2007). AT340 (2216 m) contained multiple mounds located on a topographic high. Submersible surveys of the area documented extensive carbonate substrata, large mussel beds, clumps of tubeworms and a few soft corals. GC852 (1450 m) was characterized by an elongated ridge approximately 2 km long running north to south, with vast amounts of carbonate substrata and numerous corals on the crest. Tubeworms and mussel beds were also documented at this site. Additionally, oil slicks were present on the surface and bubble streams were reported on the bottom, which may be potential mechanisms for transporting benthic material to the surface. AC601 (2340 m) differed from the other two sites, having low topography and a large brine pool. 4

16 Some carbonate substrata and a few isolated aggregations of tubeworms were present, none of which were near the brine pool. High methane concentrations in the water column were also recorded in the water column over this site (Roberts et al. 2007). Sample Collection Intense sampling of the upper 1000 m of the water column was conducted during 24 hour operations at all three sites from 9-25 August 2007; however, due to inclement weather, only minimal night sampling was conducted at AC601. A total of 173 stations (45 day, 108 night, and 20 twilight) were sampled (Table 1). Multiple gear types were utilized to adequately sample the fauna, including a Tucker trawl, Neuston net, and plankton nets, though discrete-depth Tucker trawling was emphasized. Midwater fauna were collected using a Tucker trawl (2 x 2 m, 1.59 mm mesh, 505 μm cod end.) with a plankton net (0.5 m diameter, 335 μm mesh) attached inside the Tucker trawl mouth to simultaneously sample the smaller components of the midwater fauna. Trawls were equipped with a Sea-Bird SBE39 temperature-depth recorder (TDR) attached to the upper frame bar to record time, depth, and temperature during deployment. The Tucker trawl was deployed open, and it was assumed no significant fishing occurred during deployment due to the rapid lowering, steep wire angle, and minimal forward movement of the vessel (Gartner et al., 2008; Ross et al. 2010). Upon reaching the designated depth, the trawl fished for approximately 30 min at a 2 knot (3.7 km/hr) ground speed and was triggered closed using a double trip mechanism. Actual time and depth fished for each trawl was determined post-tow using data from the TDR. TDR data were used throughout the cruise to adjust fishing strategies to achieve desired sampling depths. The mean depth for each Tucker trawl tow was calculated by averaging all depths recorded by the TDR from the start to the end of each tow. Tucker trawling 5

17 intensely sampled the upper 1000 m of the water column over the 24 hour time period at GC852 and AT340. Zooplankton samples were collected from a 1.1 x 2.4-m Neuston net (6.4-mm mesh body and 3.2-mm tail bag) or plankton nets (0.5 m diameter, 335 μm and 1.0 m diameter, 505 μm mesh) deployed at the surface and towed for minutes (Table 8.1). Particulate organic material (POM) was collected by filtering seawater through a 125-μm precombusted glass filter, and it was assumed that the majority of POM was phytoplankton derived (Kling et al., 1992). POM and zooplankton samples provided a food web base for SIA. Fishes collected were preserved in 10% seawater-formalin solution and later transferred to 50% isopropyl for storage until dietary analyses. Invertebrates were preserved in 70% ethanol, with the exception of jelly and salp specimens that were preserved in 10% seawater-formalin solution. All specimens were sorted, identified to the lowest possible taxa and measured to the nearest millimeter standard length (SL) (fishes) or total length (TL) (invertebrates). The life history stage of fishes was also documented based on the presence or absence of gonads. Fish specimens were classified as juvenile when either no gonads or immature gonads were present. Dietary Analyses Gut Content Analysis (GCA) GCA was conducted for the five most abundant midwater families (31 species) using methods outlined in Ross and Moser (1995). All abundant species collected (> 30 individuals, with the exception of stomiids) were analyzed. In order to increase sample size for Stomiidae, all stomiids, with the exception of C. sloani, were grouped together and were collectively referred to as Stomiidae. Highly abundant fish species, Cyclothone alba (n = 614), C. braueri (n = 669), C. pallida (n = 885), C. pseudopallida (n = 744), Valenciennellus tripunctulatus (n = 248), and 6

18 Notolychnus valdiviae (n = 1139), were randomly subsampled, with selected specimens spanning the collected size range of the species, encompassing all depths sampled, and including day, night and twilight samples. Selected fishes were dissected and the stomachs were removed. Stomach fullness was estimated as 0%, 5%, 25%, 50%, 75% or 100%. Empty stomachs were documented, though not included in most analyses, for day, night and twilight samples at all sites. Stomach contents were placed on a Petri dish and identified to the lowest possible taxon. Similar prey items were then piled together on a grid of 1 mm squares and flattened to a uniform height, which was measured. This height multiplied by the number of squares occupied by the food item yielded volume in mm 3. The frequency of occurrence for a prey item equaled the number of times a prey item occurred in the fish species examined divided by the total number of stomachs analyzed. The relationship between DVMs and stomach fullness was examined by plotting stomach fullness against time of day and mean sampling depth. Time of day was divided into three categories: day (0730 to 1830 hr CDT), night (2030 to 0530 hr CDT), and twilight (0530 to 0730 hr CDT, one hour on either side of average sunrise, and 1830 to 2030 hr CDT, one hour on either side of average sunset) and mean sampling depths were calculated based on Ross et al. (2010). TT tows where no mean sampling depth was calculated were excluded. Stable Isotope Analysis (SIA) Prior to specimen preservation in formalin or ethanol, samples of white muscle tissue were dissected from fishes and invertebrates and frozen. For consistency, tissue was removed from similar body regions based on the type of specimen (i.e., muscle tissue removed from the dorsal region of fishes, the caudal region of shrimps, the legs of crabs and the mantle of mollusks). When specimens were too small to extract a tissue sample, the whole body was used. Minimal contamination from other tissue types occurred as the head, scales, photophores, and entrails 7

19 were removed from specimens taken whole. For these specimens, species identification was made either prior to tissue collection, or a replicate specimen was vouchered for future identification. All collected isotope samples were dried and crushed into a powder. The majority of samples were dried to a constant weight in an oven at C. Additional samples were frozen at -80 C for 24 hours and freeze dried in a VirTis Benchtop 3.3 Vac-Freeze. I assumed there were no significant differences in isotopic ratios as a result of different drying techniques (Bosley and Wainright 1999). Tissue samples were analyzed for carbon and nitrogen isotope ratios. For each sample, μg were placed into a tin capsule and combusted in an Elemental Combustion System Model 4010 coupled to a Delta V Plus Isotope Ratio Mass Spectrometer (IRMS) via Conflo II interface at the University of North Carolina Wilmington (UNCW). POM (provided by A. Demopoulos, USGS), 49 fishes and 24 invertebrates were analyzed by IRMS at Washington State University using a Costech (Valencia, USA) elemental analyzer interfaced to a GV instruments (Manchester, UK) Isoprime IRMS. Precision of the IRMS at UNCW was verified by repeated analysis of standards USGS 40 and USGS 41, which were incorporated into each sample run. Raw delta values were corrected for linearity and normalized to known reference materials USGS 40 and USGS 41. A similar procedure was utilized at Washington State University using egg albumin powder calibrated against National Institute of Standards reference materials. Reproducibility was monitored using several organic reference standards (Fry 2007). Isotope ratios were expressed in the standard delta (δ) notation as parts per thousand ( ) according to the following equation: (R sample - R standard ) δ X = *1000 (1) R standard 8

20 where X is 13 C or 15 N and R is the corresponding ratio 13 C/ 12 C or 15 N/ 14 N. The global standards for δ 13 C and δ 15 N are Vienna PeeDee Belemnite and atmospheric nitrogen (air). A minimum of 5 samples were analyzed per fish species. Similar to GCA, the sample size of Stomiidae was increased by combining all species, with the exception of C. sloani. Similarly, 3 melamphid species (Melamphaes simus, M. typhlops, and Scopelogadus mizolepis) were grouped together to increase sample size for analyses and were referred to as Melamphaidae. Diaphus spp. included D. mollis and D. lucidus and Sternoptyx spp. included S. diaphana and S. pseudobscura. Data were examined after SIA to determine whether inorganic carbon or lipids may have significantly impacted the isotope results. According to Post et al. (2007) samples with C:N > 4 are likely affected by the presence of lipids, and inorganic carbon may be present when C:N > 3.5 or δ 13 C is highly enriched. Our results (all C:N < 4) indicated that neither lipids nor inorganic carbon significantly impacted the isotope ratios of fishes; therefore, no lipid extraction or acidification methods were utilized for fish isotope samples. In contrast, some invertebrates had high C:N values that suggested the presence of inorganic carbon in the samples. As a result, an acidification process was conducted on a subset of invertebrate samples, which included amphipods, copepods, euphausiids, jellyfish, pterapods, salps, and zooplankton, to remove any inorganic carbon. To acidify samples, 1.0 N hydrochloric acid was added one drop at a time to dried, crushed tissue samples until bubbling no longer occurred. Acidified samples were air dried for 8 hours before being re-dried in an oven at C for 24 hours. These samples were then processed by the same method utilized for nonacidifed samples (see above). As acidification can affect N values, acidified samples were reported with δ 15 N reflecting the ratio from the untreated sample and δ 13 C reflecting the acidified sample (Jacob et al. 2005; Pinnegar and Polunin 1999). 9

21 Isotope Mixing Models Isotope data were analyzed using IsoSource 3.5. IsoSource is a multisource mixing model program that calculates all possible solutions for the contribution of each prey source to a consumer s diet based on the isotopic signatures of the prey and predator (Phillips and Gregg 2003; Benstead et al. 2006). For this study, the average carbon and nitrogen values for each prey item and fish were entered into the mixing model to determine all feasible contributions. Prior to analysis, nitrogen values for consumers were corrected for trophic fractionation, set at 2, based on the trophic shift documented in my isotope data. It was assumed no trophic fractionation occurred in carbon (Demopoulos et al. 2007; France and Peters 1997). Tolerance was set at 0.2% with source increments set at 0.2%. Reported ranges represented the 1-99th percentile because the resulting ranges (minimum to maximum) are sensitive to small numbers of observations at the ends of the distribution and the 1-99th percentile range may be more robust to outliers (Philips and Gregg 2003). Trophic position analysis Data collected during GCA and SIA were used to calculate the trophic position of each individual fish based on the following two equations from Vander Zanden et al. (1997) [ V )( TP )] TP GC = ( i i + 1 (2) where TP GC is the trophic position of the fish based on gut content analysis, V i is the percent volume of a nth prey item and TP i is the trophic position of nth prey item based on data from Rybczynski et al. (2008) and TP SIA = N ( δ fish δ 1 consumer ( f ) N ) + 2 (3) 10

22 where TP SIA is the trophic position of the fish based on stable isotope analysis and f is the trophic fraction for one trophic level. Statistical Analyses Multivariate analyses were conducted on gut contents of each fish species to examine diet differences based on four factors: size, time of day, depth and location. All analyses utilized the software PRIMER-E version 6.1 (Clarke and Warwick 2001; Clarke and Gorley 2006). Factors were divided into groups as follows: SL was divided into size classes based on 5 mm increments (10-14 mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, mm, 115 mm); time of day was divided into three categories, day (0730 to 1830 hr CDT), night (2030 to 0530 hr CDT), and twilight (0530 to 0730 and 1830 to 2030 hr CTD); depth (based on mean sample depth) was divided into ranges based on 50 m increments (surface-49 m, m, m, m, m, m, m, m, m, m, m, m, m, m, m, m, m, m, m, m, m, m); and location was divided into the three sites (AT340, GC852, AC601). Organic material and animal parts (e.g., amphipod parts, copepod parts, decapod parts) were excluded prior to analyses as these food items were ambiguous and may be pieces of prey items identified to lower taxa. For each fish species, the prey item volumetric data were standardized for each individual fish by dividing the volume of each prey item by the total volume of the stomach in order to account for stomach fullness variability. Standardized volumes were then square root transformed to down weight the contributions of abundant prey items. Next, similarities among fish species were calculated using a Bray-Curtis similarity coefficient based 11

23 on each factor. The resulting similarity matrix was then subjected to a one way analysis of similarities (ANOSIM) to determine if diets were significantly different for each factor, with R>0.40 and p<0.05 used as the criteria for statistical significance. When significant differences were found using ANOSIM, a similarity percentages routine (SIMPER) was utilized to determine which prey items contributed to the dissimilarities. This process was repeated for each fish species. A similar multivariate procedure was implemented for diet comparisons among all 31 fish species, disregarding the factors size, time of day, depth and location. After constructing a Bray- Curtis similarity matrix, results were subjected to hierarchical clustering with group average linkage and non-metric multidimensional scaling (MDS). With a large sample size (n = 1327), a MDS plot can become cluttered with substantial noise in individual samples; therefore, data were averaged by PRIMER based on species prior to standardization (see above for standardization process) (Clarke and Gorley 2006). Additionally, to determine general feeding guilds, all fish species were analyzed using general prey categories (Amphipoda, Annelida, Chaetognatha, Cnidaria, Copepoda, Crustacea, Decapoda, Euphausiacea, Fish, Mollusca, Ostracoda, Salpida, and Other). For this analysis, identifiable animal parts were included in the general categories (i.e., copepod parts were included in Copepoda), but organic material and unidentifiable animal parts (e.g., crustacean parts, animal parts) were excluded. Clusters were defined at the 30% and 60% similarity levels. Statistical analyses were conducted on isotope ratios and trophic-level calculations using SigmaStat 3.4. Data were analyzed for normality and homogeneity of variance using Kolmogorov-Smirnov and Levene Median tests. One-way analysis of variance (ANOVA) was used to determine significant differences in isotopic values for primary producers, invertebrates 12

24 and fishes, with the exception of Phosichthyidae, where a t-test was used to determine differences between P. mauli and V. poweriae. A post-hoc Tukey test was used to determine specific differences among groups. Data that failed normality or equal variance tests were analyzed with ANOVA on the Ranks and the post-hoc Dunn s test. Species comparisons between sites AT340 and GC852 were analyzed using a two-way ANOVA and post-hoc Holm- Sidak test. An ANOVA on the Ranks, followed by Dunn s test, was used to determine significant differences in the trophic position based on GCA or SIA. Trophic positions calculated from GCA were compared to trophic positions calculated from SIA using a t-test; however, data that failed normality were analyzed using a Mann-Whitney rank sum test. Species with low sample sizes (n < 5) were not analyzed statistically. Regressions of δ 15 N against fish SL were conducted to determine whether ontogenetic shifts in diet occurred. Statistical significance was determined when p < Isotope data were reported with the mean ± 1 standard error. RESULTS Catch data Tucker trawling consisted of 123 tows (33 day and 90 night) from three sites (AT340, GC852, and AC601); however, minimal sampling (n = 5, night only) was conducted at AC601. The mean depth ranges sampled were: 63 to 1503 m for AT340, 21 to 1067 m for GC852, and 45 to 584 m for AC601. A total of 8,716 fishes (30 families) were collected, but 97.7% of these fishes were from five midwater fish families: Gonostomatidae (58.8%), Myctophidae (27.4%), Phosichthyidae (5.8%), Sternoptychidae (4.4%) and Stomiidae (1.3%). Gut Content Analyses (GCA) Diet composition GCA were conducted on 31 species from the five most abundant midwater fish families 13

25 (Table 2). Gut contents were analyzed from 2,989 fishes, of which 1,658 (55%) stomachs were empty. A total of 125 prey items (45 species, 37 families) were identified in the stomachs of all midwater fishes, and items were grouped into 13 general prey taxa: Amphipoda, Annelida, Chaetognatha, Cnidaria, Copepoda, Crustacea, Decapoda, Euphausiacea, Fish, Mollusca, Ostracoda, Salpida, and Other. Copepods were the dominant prey, identified in 79% of stomachs and were consumed by all species except C. sloani. The MDS ordination plot of mean prey volumes for the 31 midwater fish species defined three general feeding guilds at a 30% similarity level (Fig. 2): piscivores, large crustacean consumers, and zooplanktivores. At a 60% similarity, the piscivore guild remained unchanged, but the large crustacean consumer guild was subdivided into two subguilds, decapod-euphausiid consumer and decapod-piscivore, and the zooplanktivore guild was subdivided into three subguilds, copepod consumer, mixed zooplanktivore, and a generalist consumer (Fig. 2). The piscivore guild contained only one species, C. sloani (Fig. 2). Empty stomachs occurred in over 80% of all stomachs analyzed (Table 4). Within stomachs that contained food, six prey items (3 prey categories) were identified. Myctophidae and Bregmaceros spp. were the most important prey items in overall percent volume and frequency, and no identifiable invertebrates were documented (Table 4). Large crustacean consumers consisted of G. elongatum and Stomiidae (Fig. 2). Decapods were the dominant prey item, comprising over 70% of the identifiable prey volume of this guild. At 60% similarity, this guild was divided into two subguilds: decapod-euphausiid consumer and decapod-piscivore consumer. The decapod-euphausiid consumer subguild contained one species, G. elongatum. Empty stomachs, all from night collections, occurred in 24% of analyzed G. elongatum (Table 5). This species had 29 prey items (9 prey categories) identified (Table 5), and 14

26 while decapods and euphausiids were important prey volumetrically, copepods, particularly calanoid, were consumed more frequently (Table 5). The decapod-piscivore consumer subguild was comprised of Stomiidae. Empty stomachs occurred more frequently in this subguild and were documented in 69% of all stomiids (Table 6). The diet of Stomiidae was less variable than G. elongatum and was characterized by 11 prey items (4 prey categories) identified in the stomachs. Decapods and myctophids were the most important prey in overall percent volume and frequency for Stomiidae (Table 6). All other midwater fishes were classified as zooplanktivores, which was divided into three subguilds. The copepod consumer subguild contained C. alba, C. braueri, C. pseudopallida, V. tripunctulatus, D. mollis, and H. benoiti. Copepods comprised over 90% of the diet (Fig. 2). A high percentage (> 68%) of empty stomachs occurred in C. alba (Table 7), C. braueri (Table 8), C. pseudopallida (Table 9), and H. benoiti (Table 12), while fewer empty stomachs were documented in V. tripunctulatus (17%, Table 10) and D. mollis (9%, Table 11). Although Copepoda was the major prey category consumed in terms of volume and frequency, stomachs contained a diversity of prey (Tables 7-12), ranging from 13 prey items for H. benoiti (Table 10) to 36 prey items for V. tripunctulatus (Table 11). Pleuromamma spp. was the dominant copepod in terms of volume and frequency consumed by C. alba (Table 7), V. tripunctulatus (Table 11), and D. mollis (Table 12), whereas Aegisthus mucronatus was more important in the diets of C. braueri (Table 8). Valdiviella minor was volumetrically more important in the diet of C. pseudopallida, but Lubbockia spp. occurred more frequently (Table 9). Calanoid copepods were volumetrically important in the diets of H. benoiti, but cyclopoid copepods occurred more frequently (Table 10). 15

27 The mixed zooplanktivores subguild was defined by a general crustacean diet, with species consuming a variety of zooplankton. This subguild contained C. pallida, A. aculeatus, A. hemigymnus, P. mauli, V. poweriae, B. suborbitale, L. alatus, L. guentheri, M. affine, and N. valdiviae (Figure 2). The presence of empty stomachs was variable in this subguild, ranging from 21% of specimens containing empty stomachs (L. alatus) to 94% of specimens containing empty stomachs (C. pallida). Examination of gut contents revealed the overall diet diversity for mixed zooplanktivores was greater than copepod consumers, ranging from 10 prey items for C. pallida (Table 13) to 42 prey items for V. poweriae (Table 14). Amphipoda was more important volumetrically in the diet of C. pallida (Table 13) and M. affine (Table 15), while Conchoecinae (Ostracoda) was more important for A. aculeatus (Table 16) and A. hemigymnus (Table 17). Ostracods, particularly Conchoecinae, also occurred frequently in the stomachs of C. pallida (Table 13), A. aculeatus (Table 16), and P. mauli (Table 18), while calanoid copepods (particularly Pleuromamma spp.) occurred more frequently in the stomachs of M. affine (Table 15), A. hemigymnus (Table 17), B. suborbitale (Table 19), L. alatus (Table 20), L. guentheri (Table 21) and N. valdiviae (Table 22). Similar to copepod consumers, Pleuromamma spp. and other calanoid copepods were important prey items for P. mauli (Table 18), B. suborbitale (Table 19), L. alatus (Table 20), L. guentheri (Table 21) and N. valdiviae (Table 22); however decapods, euphausiids and ostracods also influenced their diets volumetrically. Vinciguerria poweriae exhibited a more unique diet compared to other mixed zooplanktivores, with myctophids and Candaciidae (Copepoda) dominating the diet volumetrically, but Conchoecinae and calanoid copepods occurring more frequently (Table 14). The generalist subguild contained only C. warmingii. This fish had the most variable diet of any zooplanktivore, with more non-crustacean prey consumed than any other zooplanktivore. 16

28 Empty stomachs occurred in 18% of C. warmingii, while stomach contents revealed a total of 39 prey items (13 categories, Table 23). Crustaceans comprised roughly 60% of the identifiable diet and non-crustacean prey comprised about 40% (Table 23). Fish, molluscs, and copepods were more important in overall percent volume of the diet, though copepods occurred more frequently than any other prey item (Table 23). Factors influencing diet composition Variations in diet composition due to size differences were investigated using GCA. The size range was reported for each fish species (Table 2). No significant differences in diet composition were documented based on size within species (Table 3), although the majority (79%) of fishes analyzed were juveniles. Gut contents were analyzed to determine whether time affected diet composition. Statistically, similar prey was consumed by midwater fish species regardless of the time of day (Table 3); however, some general trends were documented in regards to the prevalence of empty stomachs. Empty stomachs occurred more frequently during the day ( ) in C. sloani (Tables 4), C. pallida (Tables 13), V. poweriae (Tables 14), M. affine (Tables 15), P. mauli (Tables 18), B. suborbitale (Tables 19), L. alatus (Tables 20), and C. warmingii (Tables 23), while empty stomachs occurred more frequently in the day and twilight ( ) in C. alba (Tables 7), C. braueri (Tables 8), C. pseudopallida (Tables 9), H. benoiti (Tables 10), V. tripunctulatus (Tables 11), and D. mollis (Table 12). For G. elongatum (Tables 5), Stomiidae (Tables 6), A. aculeatus (Tables 16), A. hemigymnus (Tables 17) and L. guentheri (Tables 21), empty stomachs were documented more frequently in specimens collected at night ( ), and more specimens of N. valdiviae with empty stomachs were collected at twilight ( and , Table 22). 17

29 Diet composition was examined on horizontal and vertical spatial scales in addition to temporal scales. Comparisons among sites yielded no significant differences in diet composition (Table 3). There was also no significant difference in diet composition based on depth, with the exception of A. hemigymnus (ANOSIM, R = 0.546, p = 0.019). SIMPER analysis documented an average dissimilarity of 59.3% for A. hemigymnus collected between m compared to m. Ostracoda (32.8%), Copepoda (32.1%) and Euphausiacea (29.2%) contributed to the diet dissimilarity between these depths, with less diet variability (copepods only) documented in the stomachs of specimens collected between m. Despite the lack of significant differences temporally and spatially, migration patterns were examined to document general trends in feeding. No DVMs were documented for C. alba or C. braueri (Figure 3A-B), with more full stomachs documented at night ( ) in the mid mesopelagic range ( m). DVMs were slightly evident for C. pallida, C. pseudopallida, A. hemigymnus, and V. tripunctulatus (Figure 3C-F), with more full stomachs documented during the day ( ) in the lower mesopelagic ( m) for C. pallida (Figure 3C), more full stomachs documented at night ( ) in the mid mesopelagic range ( m) for A. hemigymnus (Figure 3E) and V. tripunctulatus (Figure 3F), and C. pseudopallida consuming prey during a 24 hour period (Figure 3D). For species that underwent DVMs, G. elongatum, A. aculeatus, P. mauli, V. poweriae, B. suborbitale, C. warmingii, D. mollis, H. benoiti, L. alatus, L. guentheri, and N. valdiviae (Fig. 3G-Q), fuller stomachs occurred more frequently at night in the epipelagic/upper mesopelagic (surface to 350 m). Myctophum affine deviated from this pattern in migrating midwater fishes, with fuller stomachs occurring more frequently at night in the mid mesopelagic (Fig. 3R). Stomiids were another exception, with C. sloani having more full stomachs at night in the mid mesopelagic ( m, Fig. 3S) and 18

30 Stomiidae having more full stomachs during the day in the lower mesopelagic ( m, Fig. 3T). Stable isotope analyses (SIA) SIA were conducted on 337 samples, collected from the Neuston net (n = 1), plankton nets (n = 41), TT (n = 274), and filtered seawater (n= 21). These samples represented 30 fish species (6 families), 10 general invertebrate taxa (Amphipoda, Cephalopoda, Chaetognatha, Cnidaria, Copeopda, Decapoda, Euphausicea, Gastropoda, Salpida, Zooplankton) and three potential carbon sources (detritus, Sargassum spp., and POM, Table 2). Spatial variations in δ 13 C and δ 15 N were examined for fishes, invertebrates and carbon sources (Table 24). No statistical comparisons were conducted on detritus (only collected at AT340), or Sargassum spp. (n < 5 at AC601 and AT340). POM sampling revealed no significant difference in δ 13 C among sites; however, samples collected at GCA852 were depleted in 15 N compared to AT340 (post-hoc Tukey test, p = 0.003). Small sample sizes of invertebrates at each site also prevented statistical spatial comparisons on all invertebrate categories except Copepoda, Decapoda and Euphausiacea (Table 24). There were no significant differences in 13 C or 15 N for Copepoda between sites GC852 and AT340. Neither Decapoda nor Euphausiacea had any significant differences in 13 C between GC852 and AT340; however, both were significantly enriched in 15 N at GC852 compared to AT340 (post-hoc Tukey test, p < 0.001). Spatial comparisons among fishes collected GC852 and AT340 were also limited by small sample sizes and only conducted on G. elongatum, A. aculeatus, V. poweriae, and L. alatus. There were no significant differences in δ 13 C within any fish species collected at GC852 or AT340. Nitrogen was significantly enriched in G. elongatum and L. alatus collected at GC852 compared to specimens collected at AT340 (Holm-Sidak, unadjusted p < 0.001, unadjusted p = 0.008), while 19

31 A. aculeatus was depleted in 15 N at GC852 compared to AT340 (Holm-Sidak, unadjusted p = 0.049) and V. poweriae had no significant differences in δ 15 N between GC852 and AT340. Valenciennellus tripunctulatus, the only fish species statistically analyzed at all three sites, was significantly enriched in 13 C at AT340 compared to V. tripunctulatus collected at GC852 and AC601 (Tukey, p = 0.018); however, there were no significant differences in δ 15 N among sites. Data were also compared across sites to evaluate non-spatial species differences in isotopes. There was a clear distinction in δ 13 C for each of the three carbon sources. Detritus was significantly enriched in 13 C compared to POM (Tukey, p < 0.001) and Sargassum spp. (Tukey, p < 0.001), while Sargassum spp. was significantly enriched in 13 C compared to POM (Tukey, p = 0.031). There were no significant differences in δ 15 N between detritus and Sargassum spp. or detritus and POM; however, Sargassum spp. was significantly depleted in 15 N compared to POM (Dunn s, p < 0.05). Examination of non-spatial differences in isotopes among invertebrate taxa was also conducted; however, most specimens were grouped into general taxa categories due to small sample sizes. Both δ 15 N and δ 13 C were similar among invertebrates with the following exceptions. Salpida was depleted in 15 N compared to all other invertebrates, although this difference was only significant compared to Chaetognatha, Gennadas valens, Acanthephyra purpurea, and Copepoda (all comparisons, Dunn s, p < 0.05). Also, Acanthephyra purpurea and Systellaspis debilis were both significantly enriched in 13 C compared to Chaetognatha, Copepoda, and Zooplankton (all comparisons, Dunn s, p < 0.05). Comparisons among three decapod species, Gennadas valens, Acanthephyra purpurea, and Systellaspis debilis, revealed that G. valens was significantly depleted in 13 C compared to A. purpurea (Tukey, p = 0.02), and 20

32 S. debilis (Tukey, p = 0.02), and A. purpurea were significantly enriched in 15 N compared to S. debilis (Dunn s, p < 0.05). Similarly, non-spatial differences in δ 13 C and δ 15 N were examined among midwater fish families and species. No significant differences in δ 13 C existed among the 6 fish families; however, Sternoptychidae was significantly enriched in 15 N compared to Phosichthyidae and Myctophidae (Dunn s, p < 0.05). Additional differences were documented among individual species within each family as follows. In Gonostomatidae, there were no significant differences in δ 13 C; however, C. pallida was significantly enriched in 15 N compared to C. alba and C. pseudopallida (Dunn s, p < 0.05). In Sternoptychidae, A. aculeatus and A. hemigymnus were enriched in 13 C compared to Sternoptyx spp. and V. tripunctulatus (all comparisons, Tukey, p < 0.05), while V. tripunctulatus was significantly enriched in 15 N compared to A. hemigymnus (Dunn s, p < 0.05). Between phosichthyid species, V. poweriae was significantly enriched in 15 N, but depleted in 13 C compared to P. mauli (t-test, p < 0.001). All stomiid species exhibited similar isotopic signatures, with no significant differences in δ 15 N or δ 13 C. Among myctophid species, M. affine was significantly depleted in 13 C compared to all other myctophids (Tukey, p < 0.05) and was also depleted in 15 N, though differences were only significant when compared to Diaphus spp., D. problematicus, and L. alatus (all comparisons, Dunn s, p < 0.05). Diaphus spp. was significantly enriched in 15 N compared to C. warmingii (Dunn s, p < 0.05) and D. problematicus was enriched in 13 C compared to L. alatus (Tukey, p < 0.05). SIA also indicated trophic relationships within the mesopelagic food web. Enrichment in 15 N was evident with increasing trophic levels, with a trophic fractionation of roughly 2, while trophic fractionation in δ 13 C was less apparent (Fig. 4). No distinct chemosynthetic signature (δ 13 C ranging from -75 to -28 ) was detected in any flora or fauna, with the δ 13 C values for all 21

33 fishes reported within the range of photosynthetic-based material. The first trophic level, representing the base of the mesopelagic food web, was comprised of POM (Fig. 4). The second trophic level, identified after applying a 2 trophic fractionation to POM, contained mostly zooplankton, such as Copepoda, Euphausiacea, and Amphipoda (Fig. 4). The third trophic level, designated by a second 2 trophic enrichment, encompassed the majority of mesopelagic fishes (Fig. 4), with one exception (M. affine), which was depleted in both 15 N and 13 C, relegating it to the second trophic level. IsoSource was used to calculate the potential contribution of each prey category to the midwater fishes (Table 25). Crustaceans were the dominant prey and were reported in the diets of all midwater fishes. Zooplankton was an important prey item for C. alba, Sternoptyx spp., V. tripunctulatus, C. sloani, and Melamphaidae, with potential contributions ranging from 18-98% of their diets. For A. aculeatus, A. hemigymnus, P. mauli, Stomiidae, D. problematicus, and L. guentheri, Decapoda was an important prey item, with potential contributions ranging from 2-84%. Non-crustacean prey items, such as Pterapoda, had contributions ranging from 2-54% of the diets of C. alba, Sternoptyx spp., V. poweriae, P. mauli, and C. warmingii, while Salpida had contributions ranging from 8-66% of the diet for P. mauli. In some cases, such as C. pallida, C. pseudopallida, G. elongatum, B. suborbitale and L. alatus, it was not possible to determine prey contributions to the diets with confidence. The lack of confidence in determining prey contributions stemmed from all prey sources having a minimal contribution of zero to the diets, with these fishes not confined within the isotopic signatures of the prey items analyzed in IsoSource. Myctophum affine deviated from all other midwater fishes, with no solutions generated for diet contribution based on the zooplankton and POM analyzed due to the depleted 13 C reported; however, solutions were generated when the average isotopic signature of 22

34 chemosynthetic material (based on published literature) was included in the IsoSource analysis. Minimal contributions of chemosynthetic material ranged from 22-30% of the diet for M. affine; however, this did not necessarily indicate consumption of chemosynthetic material because values were based on averages and the isotopic signature of M. affine was within the range of photosynthetic material. Ontogenetic shifts were investigated for Cyclothone alba, C. pallida, C. pseudopallida, G. elongatum, A. aculeatus, A. hemigymnus, Sternoptyx spp., V. tripunctulatus, P. mauli, V. poweriae, C. sloani, Stomiidae, B. suborbitale, C. warmingii, Diaphus spp., D. problematicus, L. alatus, L. guentheri, M. affine, and Melamphaidae (Fig 5A-T) by examining the relationships between δ 15 N and SL. Positive relationships between δ 15 N and SL were identified in nineteen of the twenty species analyzed; however, significant relationships were identified in C. pseudopallida (R 2 = 0.736, p = 0.002), G. elongatum (R 2 = 0.618, p = 0.002), V. poweriae (R 2 = 0.614, p < 0.001), C. sloani (R 2 = 0.852, p = 0.009), D. problematicus (R 2 = 0.738, p = 0.013), Diaphus spp. (R 2 = 0.838, p = 0.029) and Melamphaidae (R 2 = 0.687, p = 0.003). One significant negative relationship was also identified in M. affine (Fig. 3S), with lower δ 15 N documented in larger individuals (R 2 = 0.408, p = 0.047). Trophic position calculations Trophic position calculations for the midwater fishes varied by the type of analysis (GCA versus SIA). Using data from GCA, the calculated trophic positions among fish species ranged from 2.90 (C. warmingii) to 4.00 (Stomiidae, Table 26). Significant differences among these trophic positions only occurred between C. sloani and C. braueri (Dunn s, p < 0.05) and Stomiidae and C. braueri (Dunn s, p < 0.05), with the stomiids occupying a higher trophic position. For isotope data, the calculated trophic positions of midwater fishes had a broader range 23

35 than those derived from GCA. Trophic positions from SIA ranged from 1.19 (M. affine) to 3.96 (C. pallida), and more significant differences in trophic positions were documented among fish species. The myctophid M. affine occupied a significantly lower trophic position than C. pallida, A. aculeatus, Sternoptyx spp., V. tripunctulatus, Diaphus spp., and Melamphaidae, while C. warmingii occupied a significantly lower trophic position than C. pallida, V. tripunctulatus and Diaphus spp. (All comparisons, Dunn s, p < 0.05). Also, P. mauli occupied a significantly lower trophic position than C. pallida (Dunn s, p < 0.05), A. aculeatus (Dunn s, p < 0.05), V. tripunctulatus (Dunn s, p < 0.05), and Diaphus spp. (Dunn s, p < 0.05), while V. tripunctulatus occupied a significantly higher trophic position than B. suborbitale (Dunn s, p < 0.05) and L. guentheri (Dunn s, p < 0.05). Trophic positions of midwater fishes calculated from SIA data were significantly lower than trophic positions calculated from gut content data for C. alba (Mann-Whitney, p < 0.001), C. pseudopallida (p < 0.001), P. mauli (p < 0.001), V. poweriae (p = 0.025), C. sloani (p < 0.001), Stomiidae (p = 0.022), B. suborbitale (p < 0.001), C. warmingii (t-test, p < 0.001), L. guentheri (Mann-Whitney, p < 0.001), and M. affine (Mann-Whitney, p < 0.001). In contrast, C. pallida (Mann-Whitney, p = 0.022) and V. tripunctulatus, (p < 0.001) occupied significantly higher trophic positions according to data from SIA than GCA. There were no significant differences between trophic positions calculated from GCA and SIA for G. elongatum, A. aculeatus, A. hemigymnus, Diaphus spp. and L. alatus. DISCUSSION Diet Composition Zooplankton was the dominant prey for midwater fishes. Based on SIA, all species, with the exception of M. affine, were one trophic level above zooplankton. Additionally, copepods, particularly Pleuromamma spp., were prevalent in the stomachs of all midwater fishes except C. 24

36 sloani. This prevalence of zooplankton in the diets of midwater fishes, which was support with SIA, suggested midwater fishes may be competing for zooplankton prey; however, a more detailed examination of diet composition using GCA revealed three feeding guilds within midwater fishes, similar to results from Gartner et al. (1997). Chauliodus sloani occupied a different guild than all other midwater fishes, with only fishes documented in the stomachs. Physical adaptations, such as large curved teeth, an expansive oral cavity and a lack of ossification in the anterior vertebrate, which allow the skull to move upward and back (Borodulina 1972), make it easier for C. sloani to capture larger prey like myctophids and Bregmaceros spp. The high number of empty stomachs in C. sloani may indicate that foraging was not always successful in the epipelagic zone (Sutton and Hopkins 1996; present study). Sutton (2005) suggested zooplankton may be consumed by C. sloani to sustain energetic needs between successful feeding on larger prey and crustaceans were previously documented in the stomachs of C. sloani in the eastern GOM (Hopkins et al. 1996; Sutton and Hopkins 1996), Arabian Sea (Butler et al. 2001) and off Hawaii (Clarke 1982). IsoSource also supported this concept of zooplankton consumption, with 48-90% of the diet of C. sloani comprised of zooplankton. This relationship between foraging success and zooplankton consumption may also explain ontogenetic diet shifts documented in C. sloani using SIA. Roe and Badcock (1984) reported that crustaceans, particularly euphausiids, were consumed more by smaller (< 120 mm) specimens of C. sloani (Roe and Badcock 1984), which were likely less efficient at capturing fishes. Overall, the incorporation of zooplankton suggested that despite occupying the piscivore guild, C. sloani may feed more similar to other midwater fishes than previously thought. Similar to C. sloani, Stomiidae occupied a different guild than the majority of midwater fishes. Fishes, particularly myctophids, were consumed by Stomiidae, revealing some trophic 25

37 similarity to C. sloani; however, Stomiidae was classified as a large crustacean consumer due to the dominance of decapods in the diet. Placement in this guild was further supported by IsoSource, with decapods comprising 2-58% of the diets. Previous literature documented decapods, euphausiids and copepods in the stomachs of stomiid species, such as Astronesthes, Photostomias and Malacosteus; however, other stomiids, such as Idiacanthus and Stomias consumed fishes, (Clarke 1982; Hopkins et al. 1996; Sutton and Hopkins 1996; Sutton 2005; present study). Differences in diet composition among stomiid species suggested that guild classification for Stomiidae was not robust because guild placement for Stomiidae was dependent on the species grouped together for analyses; therefore, if more piscivorous stomiids were analyzed in this study, Stomiidae would occupy a niche more similar to C. sloani than G. elongatum. Even though guild placement was variable, the overall trophic position of Stomiidae remained unchanged because large crustaceans, such as the decapod G. valens, consumed zooplankton (Hopkins et al. 1994), similar to myctophids, such as D. mollis; therefore, regardless of whether Stomiidae consumed fishes or large crustaceans, Stomiidae remained a tertiary consumer. Gonostoma elongatum was classified in the same guild as Stomiidae, with decapods documented as the dominant prey. In addition to consuming large crustaceans, G. elongatum frequently incorporated smaller zooplankton, such as the copepod Pleuromamma spp., into its diet. This was similar to previous findings in the eastern GOM (Lancraft et al. 1988; Hopkins et al. 1996) and was supported by SIA. Clarke (1982) suggested G. elongatum was a zooplanktivore but it consumed large crustaceans because it reached larger sizes than other zooplanktivores. This, along with the previously mentioned studies, suggested diets shifted with ontogeny; however, GCA reported G. elongatum consumed similar prey regardless of size. It 26

38 was possible that larger G. elongatum consumed similar, but trophically higher prey specimens. Ontogenetic diet changes were documented in prey like euphausiids (Gurney et al. 2001), and could reveal ontogenetic diet shifts in G. elongatum with isotope data, which was documented in this study. These diet changes can alter the trophic position of G. elongatum, with larger specimens that consumed predatory prey, such as chaetognaths and Gennada valens, reported as trophically similar to piscivorous stomiiids, while smaller specimens were trophically similar to zooplanktivores. As a result, G. elongatum may occupy two different trophic guilds despite consuming similar taxa. The majority of midwater fishes were classified in the zooplanktivore guild. Isotope data also indicated a zooplanktivorous diet for midwater fishes, as fish species occupied roughly one trophic level above zooplankton. Overall, Pleuromamma spp. was the dominant prey consumed by the majority of midwater fishes (Hopkins and Baird, 1981; Hopkins et al., 1996; Sutton et al., 1998; present study), and the prevalence of Pleuromamma spp. in stomachs was attributed to its wide distribution in the upper 1000 m (Deevey and Brooks 1977). Despite the prevalence of Pleuromamma spp. in midwater fishes stomachs, the inclusion of other zooplankton species subdivided the zooplanktivore guild into copepod consumers, mixed zooplanktivores and generalists, which may reduce competitive pressures on prey among the zooplanktivores. Copepods were the dominant prey for C. alba, C. braueri, C. pseudopallida, V. tripunctulatus, D. mollis and H. benoiti, supporting previous reports (Hopkins and Baird 1981; Hopkins et al. 1996). Although over 90% of the diet contained copepods, examination of the composition and vertical distribution of the copepod prey (Pearcy et al. 1979), suggested competitive pressure on copepods was not high as species were not consuming the same copepod species. The deepwater copepod Aegisthus mucronatus, documented 500 to 1500 m (Deevy and 27

39 Brooks 1977; Razouls et al ) was only reported in the stomachs of C. braueri, C. pseudopallida and V. tripunctulatus, suggesting these species fed at a deeper depths and indicating vertical space as a factor contributing to diet composition. Interestingly, shallower water copepod species, such as Lubbockia aculeata (0-500 m, Deevey and Brooks 1977) and Corycaeus spp. (0-300 m, Roehr and Moore 1965), were also present in the stomachs of C. braueri, C. pseudopallida and V. tripunctulatus. This indicated DVMs, which although not reported in C. braueri. (Badcock and Merrett 1977; Miya and Nemoto 1987; present study) was documented in C. pseudopallida and V. tripunctulatus (Ross et al. 2010; present study). Even though depth appeared to have some influence in prey selection, according to GCA, depth did not significantly affect prey preferences, with species generally consuming the same prey at all depths. Size could have influenced this depth related diet composition, as larger individuals of a fish species often occupied deeper depths (Hopkins and Sutton 1998) and therefore consumed deepwater copepods, while smaller midwater fishes that occupied shallower depths consumed shallow water copepod species. Unfortunately, gut content data did not support diet variation by growth, and isotope data only identified ontogenetic diet shifts in C. pseudopallida and Diaphus spp. Therefore, other parameters must be investigated to determine what other factors influence prey selection within copepod consumers. Differentiation in the diets of copepod consumers can also reduce competition for copepod prey. Cyclothone alba occupied the mid mesopelagic, similar to other Cyclothone spp., but C. alba consumed decapods, in addition to copepods, thus utilizing different prey resources. Similarly, H. benoiti, D. mollis, and V. tripunctulatus occupied overlapping vertical depths, but only the myctophids incorporated decapods into their diets. Additionally, D. mollis consumed a variety of non-crustacean prey, such as fish, chaetognaths and mollusks, which separated it from 28

40 H. benoiti. These variations may reduce competition for copepods within the copepod consumer subguild, particularly as similar vertical space was occupied. The majority of zooplanktivorous midwater fishes were classified as mixed zooplanktivores, which was supported by isotope data. Calanoid copepods, particularly Pleuromamma, were an important diet component for C. pallida, A. aculeatus, A. hemigymnus, P. mauli, V. poweriae, B. suborbitale, L. alatus, L. guentheri, and N. valdiviae, but ostracods, euphausiids, and amphipods were also incorporated (Hopkins and Baird 1981; Hopkins and Baird 1985a; Hopkins et al. 1996; Sutton et al. 1998; present study), which may reduce competition for Copepoda by consuming different compositions of zooplankton. Argyropelecus spp. ate a mixture of copepods, ostracods, amphipods and euphausiids, which agreed with previous studies (Hopkins and Baird 1981; Hopkins and Baird 1985a; Sutton et al. 1998); however, A. aculeatus also targeted noncrustacean prey, particularly mollusks, while A. hemigymnus targeted only ostracods and copepods (Hopkins and Baird 1985a; Kawaguchi and Mauchline 1987; present study). For C. pallida and M. affine, amphipods were selectively consumed; however, previous studies only supported this selectivity for M. affine (Hopkins and Gartner 1992), as C. pallida was previously known to target ostracods (Burghart et al. 2010). Lampanyctus alatus and L. guentheri targeted halocyprid ostracods, though euphausiids were considered a dominate prey item in previous studies (Hopkins and Baird 1985b; Hopkins et al. 1996). The importance of euphausiids in the diets of L. alatus and L. guentheri increased with size (Hopkins and Baird 1985b; Hopkins and Gartner 1992), and the differences in diet composition among these studies were attributed to the majority (84%) of specimens in this study being juveniles (< 30 mm). The prevalence of juveniles also explained the lack of ontogenetic diet shifts in GCA. Other species, such as V. poweriae, B. suborbitale, and D. mollis, occasionally incorporated fishes into their diets, which 29

41 reduced competition for copepods, amphipods, ostracods and euphausiids as prey. This also explained the enriched δ 15 N documented in V. poweriae compared to P. mauli, which did not consumed any fishes. Gelatinous prey, such as salps and mollusks, also played a role in the diets of mixed zooplanktivores, though these prey were often underestimated since they were digested more quickly than crustaceans (Gartner et al. 1997). Ceratoscopelus warmingii also had a mixed zooplankton diet, with almost 40 different prey items identified in its stomach; however, C. warmingii was classified into its own subguild because almost 40% of the diet contained non-crustacean prey, which was supported by IsoSource. This high diet diversity in C. warmingii was previously documented in Hopkins and Baird (1975) and Hopkins et al. (1996), with Robinson (1984) also noting C. warmingii as an occasional herbivore. By establishing a generalist feeding strategy, C. warmingii can occupy a unique niche, despite being restricted by a narrow spatial and temporal feeding pattern as documented in the majority of zooplanktivores (Robinson 1984). Spatial and Temporal influences on diet Resource partitioning in the midwater community was previously reported by Hopkins and Sutton (1998) using parameters such as depth, time and size. Although size, depth, location and time of day did not affect prey preferences for individual species, it was evident these parameters influenced the trophic structure of the midwater community (Hopkins and Sutton 1998). In the piscivore guild, competition for fish prey may be reduced through the utilization of vertical space even though all specimens of C. sloani occupied the same feeding guild. Chauliodus sloani occupied the mid mesopelagic, which contained fewer midwater fish species than the upper mesopelagic for C. sloani to compete with for fish prey. Additionally, asynchronous migrations previously documented in C. sloani suggested only the hungry portion 30

42 of stomiids migrate to the epipelagic (Sutton and Hopkins et al. 1996). This migration pattern was also apparent in this study and utilization of vertical space in this manner allowed C. sloani to effectively partition resources, even with C. sloani occupying the same guild and habitat as other midwater fish species. Competition among large crustacean consumers for decapods was also influenced by vertical space. In general, Stomiidae occupied the lower mesopelagic zone during the day, while G. elongatum occupied the mid mesopelagic. This spatial variation suggested these species may not be competing for large crustaceans, as G. elongatum and Stomiidae may consume prey at different depths. Additionally, these crustacean prey were also vertically distributed (Hopkins and Sutton 1998) and therefore may be consumed at different depths. Utilization of vertical space for migrations was also used differently with this guild, with Stomiidae undergoing asynchronous migrations (Sutton and Hopkins et al. 1996; Kenaley 2008; present study), thereby reducing predation pressures on large crustaceans since all stomiids did not migrate to the epipelagic at night, while G. elongatum underwent DVMs, with the majority of specimens migrating to the upper mesopelagic/epipelagic to feed (Lancraft et al. 1988; present study). This was similar to the migration pattern documented in zooplanktivores, like myctophids (Hopkins and Gartner 1992), suggesting G. elongatum may be more similar to zooplanktivores than to Stomiidae. Despite the lack of DVMs in C. alba and C. braueri, utilization of vertical space may influence other copepod consumers, like D. mollis and H. benoiti. These myctophids migrated to the surface at night, feeding at shallower depths than the other copepod consumers. Additionally, the DVMs undertaken by these fishes followed the migration of copepod prey, such as Pleuromamma spp. (Pusch et al. 2004), which enabled these myctophid species to feed on dense 31

43 prey populations in the epipelagic. This use of vertical space ensured D. mollis and H. benoiti did not have to compete with other copepod consumers for their copepod prey sources. Examination of time, though not significant within any species emphasized a general trend for feeding at night. Despite the apparent preference for feeding at night, all species, except M. affine, occasionally consumed prey during the day. Feeding spread across a 24 hour period was previously documented in myctophids and sternoptychids (Merrett and Roe 1974; Clarke 1978; Pusch et al. 2001) and can enhance resource partitioning (Hopkins and Sutton 1998) as species have less restrictions. Additional insights with SIA Site differences Spatial variation due to the complex bottom topography and hydrography in the GOM was hypothesized to affect diet composition in midwater fishes, particularly as diet variations were previously attributed to locality in myctophids (Pakhomov et al. 1996; Pusch et al. 2004). However, GCA documented similar feeding among sites, which was also supported by previous findings in the eastern GOM (Hopkins et al. 1996). In contrast, spatial variations were documented with SIA and indicated potential changes in the prey species consumed. Carbon values for phytoplankton were similar among sites; however, V. tripunctulatus was enriched δ 13 C at AT340. Warm core rings, such as the one present during sampling at AT340 (Ross et al. 2010), can change zooplankton biomass by increasing diatom productivity and thereby alter isotopic composition in POM and zooplankton (Waite et al. 2007). Since enriched values reflected the assimilated prey consumed by V. tripunctulatus at an earlier time than it was possible that POM and invertebrate samples would also reflect enriched δ 13 C if collected after the ring moved out of the sampling area. This concept was also true for invertebrates. Although 32

44 δ 13 C was similar, both decapods and euphausiids were enriched in δ 15 N at GC852 compared to AT340, which suggested these species may have consumed trophically higher organisms or that the zooplankton biomass present during sampling was different from the zooplankton present before the warm-core ring. Unfortunately as sampling was only conducted during the presence of the warm core ring, it was not possible to confirm this notion. In contrast, G. elongatum, A. aculeatus, and L. alatus were enriched in δ 15 N at GC852 compared to AT340. Generally, an increase δ 15 N suggested an increase in trophic level (Fry 1988); however, the overall trophic structure was similar at both sites, with specimens documented as zooplanktivores, which was confirmed with GCA. Ontogenetic diet shifts could also explain this difference, but an ontogenetic diet shift was only documented in G. elongatum. The location of the study sites may also influence isotope values of these fishes. The Mississippi River affected isotope values in the northwestern GOM, with riverine sources causing enriched δ 15 N in king mackerel (Roelke and Cifuentes 1997). If the Mississippi River did cause this spatial difference, its effects would be apparent in more taxa; however, this was not the case. Therefore, diet composition may was the most likely cause for enriched 15 N in G. elongatum, A. aculeatus, and L. alatus. Soft bodied prey, such as chaetognaths and mollusks, may be consumed more at GC852, but, as previously stated, were underestimated in GCA due to faster digestive rates of soft bodied prey. Diet variations SIA implied M. affine utilized a generalist feeding strategy similar to C. warmingii. Myctophum affine was previously reported to primarily consume crustaceans (Hopkins and Sutton 1998), placing it on the third trophic level; however, the low δ 15 N values suggested M. affine occupied the second trophic level. Low δ 15 N values in M. affine may result from M. affine 33

45 ingesting Trichodesium, a cyanobacterium with global distribution that can undergo extensive blooms and supply new nitrogen to areas in which it is found (Holl et al. 2007). Previous literature reported Trichodesmium depleting δ 15 N values of POM (Montoya et al. 2002), a signal that may be passed up the food chain. It was also possible that M. affine was herbivorous, although GCA revealed only minimal amounts of phytoplankton in the stomach. Additionally, M. affine may consume δ 15 N depleted prey items, such as salps, which would place M. affine in a niche more closely related to C. warmingii. Methodology Trophic position calculations provided a characterization of the trophic structure of midwater fishes by using GCA and SIA (Vander Zanden et al. 1997; Woodward and Hildrew 2002; Rybcynski et al. 2008) and enabled a quantitative comparison between GCA and SIA. Of the 17 midwater fish species compared, differences between methods were significant in 12 species, highlighting the importance of incorporating multiple techniques to discern trophic relationships among midwater fishes (Vander Zanden et al. 1997; Woodward and Hildrew 2002; Rybcynski et al. 2008). In most cases, SIA designated fish species at a trophically lower position than GCA. It was possible that nitrogen-depleted gelatinous prey, which are quickly digested and often unidentifiable (Gartner et al. 1997), were consumed more frequently than previously documented and play a more significant role in the diets of midwater fishes. Also, if midwater fishes consumed trophically higher prey items, like fish, on occasion then GCA-based trophic positions would be greater than SIA-based because rare prey are masked by the continuous presence of trophically low prey items, like copepods, but its presence in the gut would increase the GCAbased trophic position. 34

46 Utilization of both methods allowed inferences to be made when limitations occur in one method, such as limited data from empty stomachs or documenting only generalized prey categories in the diets. For example, all Cyclothone spp. were zooplanktivores, however, C. pallida was enriched in 15 N compared to other Cyclothone spp. GCA revealed that C. pallida consumed mixed zooplankton as opposed to targeting copepods, as documented in C. alba, C. braueri and C. pseudopallida. This difference in prey composition were also evident for sternoptychids, with the mixed zooplanktivore A. hemigymnus having enriched 15 N compared to the copepod consumer V. tripunctulatus. Although these differences were documented using SIA, SIA only provides a general overview of the diet and GCA was needed to identify the subtle difference in diets for species within the trophic guild (Rybczynski et al. 2008). Another advantage of utilizing SIA was the ability to determine diet information if few specimens are collected or if GCA provided little data. Sternoptyx spp. and Melamphaidae were analyzed using SIA and were placed in the zooplanktivore guild despite low sample sizes and examination of previous literature (Hopkins and Baird 1985a; Hopkins et al. 1996) supported these results. Interesting Note SIA indicated that chemosynthetic energy did not significantly influence the midwater community. This does not however prove that chemosynthetic energy had no influence, but rather the extent of influence was below a measurable degree using the above methods. Additionally, one G. elongatum, captured in the benthic otter trawl, was significantly depleted 13 C (-25 ) indicating the assimilation of chemosynthetic material. This specimen, though collected in the benthic otter trawl, may undergo DVM and interact with other species in the water column. It was also possible that midwater fishes were aggregating on the bottom and exploiting food resources, as previously documented in the southeastern US (Gartner et al. 2008) 35

47 and documented in benthic fauna near seeps (MacAvoy 2002). Unfortunately, midwater sampling conducted in this study did not extend to the bottom and therefore would have avoided capture. CONCLUSIONS 1) The basic trophic structure of midwater fishes in the north-central GOM was classified in to three guilds: piscivore, large crustacean consumer and zooplanktivore; however zooplankton was a common prey source and documented in the stomachs of all species except C. sloani. 2) Although size, depth, time and location did not significant affect diet composition, size and depth may influence prey selection, as the majority of specimens analyzed were juveniles and different species occupied different depth ranges. 3) DVMs were apparent in many species, with species following prey to the epipelagic at night; however, feeding was not limited to the epipelagic or to night, which may help reduce competitive pressure for zooplankton. 4) GCA and SIA complemented each other and differences between methods highlighted the importance of utilizing both to discern trophic structure accurately because SIA documented only general feeding patterns, while GCA provided details on the prey that assist with determining feeding guilds in the midwater fish community. 5) Utilization of chemosynthetic energy sources was not documented in the midwater fish community, though this did not prove chemosynthetic cold seep community had no influence on midwater fishes, as influences may be minor and undetected by the methods utilized in this study. 36

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59 Table 1. Surface and midwater stations sampled over three cold seep sites (AT340, GC852, and AC601) (see Fig.1) in the Gulf of Mexico (9-25 August 2007). TT = Tucker trawl including plankton net inside Tucker trawl, PN 1 = 0.5 m dia. plankton net, PN 2 = 1 m dia. plankton net, NN = Neuston net, 5 GB = 5 gallon bucket for POM samples, D = day (0730 to 1830 hr CDT), N = night (2030 to 0530 hr CDT), TW = twilight (0530 to 0730 and 1830 to 2030 hr CDT). * = maximum depths sampled for non discrete tows (TT did not close and fished to surface). Blanks in depth columns indicated TDR did not record any data. Station Date Site Gear Time Start Latitude Start Longitude End Latitude End Longitude Mean Depth Sampled (m) CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 5 GB N CH Aug-07 GC852 TT N CH Aug-07 GC852 5 GB N CH Aug-07 GC852 TT N * CH Aug-07 GC852 TT N CH Aug-07 GC852 PN 1 N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N * CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N * CH Aug-07 GC852 PN 1 N CH Aug-07 GC852 PN 1 D CH Aug-07 GC852 TT D CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT N * CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N

60 Table 1 cont. CH Aug-07 GC852 5 GB N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 PN 1 N CH Aug-07 GC852 PN 1 D CH Aug-07 GC852 NN D CH Aug-07 GC852 TT D CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT N * CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 5 GB N CH Aug-07 GC852 PN 1 N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 5 GB N CH Aug-07 GC852 TT N CH Aug-07 GC852 PN 1 N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N

61 Table 1 cont. CH Aug-07 GC852 TT N CH Aug-07 GC852 NN D CH Aug-07 GC852 5 GB D CH Aug-07 GC852 NN D CH Aug-07 GC852 5 GB D CH Aug-07 GC852 TT D CH Aug-07 GC852 5 GB D CH Aug-07 GC852 TT D CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT D CH Aug-07 GC852 TT D CH Aug-07 GC852 TT D CH Aug-07 GC852 TT D CH Aug-07 GC852 TT D * CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT D CH Aug-07 GC852 TT D CH Aug-07 GC852 TT D

62 Table 1 cont. CH Aug-07 GC852 TT D CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT TW CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 GC852 TT N CH Aug-07 AC601 TT N CH Aug-07 AC601 TT N CH Aug-07 AC601 PN 1 N CH Aug-07 AC601 5 GB N CH Aug-07 AC601 TT N CH Aug-07 AC601 5 GB N CH Aug-07 AC601 TT N CH Aug-07 AC601 5 GB N CH Aug-07 AC601 TT N CH Aug-07 AC601 5 GB D CH Aug-07 AC601 5 GB D CH Aug-07 AT340 TT TW CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N

63 Table 1 cont. CH Aug-07 AT340 5 GB N CH Aug-07 AT340 5 GB D CH Aug-07 AT340 TT D CH Aug-07 AT340 5 GB D CH Aug-07 AT340 TT TW CH Aug-07 AT340 5 GB N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT TW CH Aug-07 AT340 TT D CH Aug-07 AT340 PN 1 D CH Aug-07 AT340 TT D CH Aug-07 AT340 TT D CH Aug-07 AT340 PN 1 D CH Aug-07 AT340 TT D CH Aug-07 AT340 TT TW * CH Aug-07 AT340 TT N CH Aug-07 AT340 5 GB N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N * CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 5 GB N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT TW CH Aug-07 AT340 PN 1 N CH Aug-07 AT340 TT TW CH Aug-07 AT340 TT D CH Aug-07 AT340 TT D

64 Table 1 cont. CH Aug-07 AT340 5 GB D CH Aug-07 AT340 TT D CH Aug-07 AT340 TT D CH Aug-07 AT340 PN 1 D CH Aug-07 AT340 TT D CH Aug-07 AT340 5 GB D CH Aug-07 AT340 TT D CH Aug-07 AT340 PN 2 D CH Aug-07 AT340 TT D CH Aug-07 AT340 TT TW CH Aug-07 AT340 TT N CH Aug-07 AT340 PN 2 N CH Aug-07 AT340 TT N CH Aug-07 AT340 PN 2 N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 5 GB N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT TW CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 PN 2 N CH Aug-07 AT340 TT N CH Aug-07 AT340 PN 2 N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 PN 2 N

65 Table 1 cont. CH Aug-07 AT340 TT D * CH Aug-07 AT340 TT D CH Aug-07 AT340 TT TW CH Aug-07 AT340 TT TW CH Aug-07 AT340 PN 2 N CH Aug-07 AT340 TT N CH Aug-07 AT340 PN 2 N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 TT N CH Aug-07 AT340 PN 2 N

66 Table 2. The total number of all midwater fishes, invertebrates and autotrophs examined in dietary analyses from the North-central GOM. GCA = gut content analysis. SIA = stable isotope analysis, SL range = standard length size range for fish species (mm). Fish species marked with an * were grouped at family level for all analyses. Fish species marked with ^ were grouped at genera for stable isotope analyses. Species GCA SIA SL range FISH Gonostomatidae Cyclothone acclinidens 1 46 Cyclothone alba Cyclothone braueri Cyclothone pallida Cyclothone pseudopallida Gonostoma elongatum Sternoptychidae Argyropelecus aculeatus Argyropelecus hemigymnus Sternoptyx diaphana^ Sternoptyx pseudobscura^ Valenciennellus tripunctulatus Phosichthyidae Pollichthys mauli Vinciguerria poweriae Stomiidae Astronesthes macropogon* 2 21, 34 Astronesthes similus* Bathophilus longipinnis* Bathophilus pawneei* 1 31 Chauliodus sloani Eustomias lipochirus* Eustomias schmidti* Leptostomias bilobatus* Melanostomias biseriatus* 1 40 Melanostomias valdiviae* 1 31 Photonectes margarita* Photostomias guernei* Stomias affinis* Stomias longibarbatus* 1 86 Myctophidae Benthosema suborbitale Ceratoscopelus warmingii

67 Table 2 cont. Diaphus lucidus^ 2 27, 67 Diaphus mollis^ Diaphus problematicus Hygophum benoiti Lampanyctus alatus Lepidophanes guentheri Myctophum affine Notolychnus valdiviae Melamphaidae Melamphaes simus* Melamphaes typhlops* 2 22, 25 Scopelogadus mizolepis* 1 24 AMPHIPODA Phrosinidae Anchylomera blossevillei 1 Platyscelidae 1 Platyscelus sp. 2 Pronoidae Parapronoe sp. 1 CEPHALOPODA Bolitaenidae Japetella diaphana 1 Enoploteuthidae Ancistrocheirus lesuerii 1 Histioteuthidae Stigmatoteuthis arcturi 3 CHAETOGNATHA 5 CNIDARIA Rhopalonematidae Colobonema sericeum 1 Atollidae 1 Atolla vanhoeffeni 1 COPEPODA 6 Megacalanidae Bathycalanus princeps 4 Pontellidae Labidocera sp. 2 DECAPODA Benthesicymidae Gennadas valens 14 56

68 Table 2 cont. Oplophoridae Acanthephyra purpurea 5 Systellaspis debilis 7 Sergestidae Sergia sp. 1 EUPHAUSIACEA 11 Euphausiidae Nematoscelis megalops 3 Thysanopoda sp. 2 Thysanopoda tricuspida 2 GASTROPODA Cavoliniidae Cavolinia tridentata 1 Diacavolinia sp. 2 SALPIDA Salpidae Salpa cylindrica 4 Salpa sp. 6 ZOOPLANKTON 11 AUTOTROPH Sargassaceae Sargassum fluitans 2 Sargassum sp. 10 Phytoplankton 21 Detritus 5 57

69 Table 3. Results of ANOSIM comparing effects of size, time of day, depth and location on the general prey categories consumed for each fish species. Differences are considered significant when R > 0.40 and p < Size Time of day Depth Location Significant Species Global R p Global R p Global R p Global R p differences Argyropelecus aculeatus No Argyropelecus hemigymnus Depth Benthosema suborbitale No Ceratoscopleus warmingii No Cyclothone alba No Cyclothone braueri No Cyclothone pallida No Cyclothone pseudopallida No Diaphus mollis No Gonostoma elongatum No Hygophum benoiti No Lampanyctus alatus No Lepidophanes guentheri No Myctophum affine No Notolychnus valdiviae No Pollichthys mauli No Stomiidae spp No Valenciennellus tripunctulatus No Vinciguerria poweriae No 58

70 Table 4. Percent volume and frequency of prey items consumed by Chauliodus sloani collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 2 n = 8 n = 37 n = 2 n = 5 n = 7 n = 2 E = 2 E = 7 E = 30 E = 1 E = 5 E = 6 E = 1 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F FISH Bregmaceros spp Myctophidae Unidentified fish parts CRUSTACEA < Unidentified crustacean parts < OTHER Organic material Unidentified animal parts

71 Table 5. Percent volume and frequency of prey items consumed by Gonostoma elongatum collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 0 n = 2 n = 28 n = 3 n = 3 n = 42 n = 10 E = 0 E = 0 E = 11 E = 0 E = 0 E = 10 E = 0 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda Scina pusilla CHAETOGNATHA Heterokrohnia sp CNIDARIA Cnidaria COPEPODA Aetideus acutus < Calanoida Candacia longimana Copepoda Corycaeus furcifer < Corycaeus sp < Eucalanidae < Gaetanus pileatus Haloptilus sp Pareucalanus attenuatus Pleuromamma xiphias Rhincalanus cornutus Temora stylifera <

72 Table 5 cont. Unidentified copepod parts CRUSTACEA Unidentified crustacean parts DECAPODA Decapoda EUPHAUSIACEA Euphausiidae Thysanoessa sp Thysanopoda sp MYSIDICEA Lophogastridae OSTRACODA Halocyprididae < Myodocopida Ostracoda < OTHER Organic material Unidentified animal parts

73 Table 6. Percent volume and frequency of prey items consumed by Stomiidae collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 1 n = 5 n = 13 n = 3 n = 0 n = 8 n = 6 E = 1 E = 2 E = 9 E = 2 E = 0 E = 6 E = 5 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F COPEPODA Oncaea sp Unidentified copepod parts DECAPODA Decapoda Penaeidae FISH Diaphus mollis Myctophidae Unidentified fish parts OTHER Invertebrate < Nematoda Organic material Unidentified animal parts

74 Table 7. Percent volume and frequency of prey items consumed by Cyclothone alba collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 2 n = 37 n = 97 n = 41 n = 58 n = 34 n = 21 E = 2 E = 26 E = 66 E = 28 E = 39 E = 22 E = 15 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F COPEPODA Aetideidae Calanoida Copepoda Cyclopoida Euchirella curticauda Euchirella sp Heterorhabdidae Lubbockia aculeata Pleuromamma robusta Pleuromamma sp Pleuromamma xiphias Poecilostomatoida Unidentified copepod parts CRUSTACEA Unidentified crustacean parts DECAPODA Decapoda OSTRACODA Halocyprididae Myodocopida

75 Table 7 cont. OTHER Organic material Nematoda Unidentified animal parts

76 Table 8. Percent volume and frequency of prey items consumed by Cyclothone braueri collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 2 n = 18 n = 100 n = 49 n = 55 n = 58 n = 37 E = 2 E = 14 E = 77 E = 34 E = 47 E = 41 E = 35 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F COPEPODA Aegisthus mucronatus Calanoida Copepoda < Corycaeus sp Cyclopoida Lubbockia aculeata Miracia efferata Pleuromamma sp Unidentified copepod parts CRUSTACEA Unidentified crustacean parts OSTRACODA Conchoecinae Ostracoda Unidentified ostracod parts OTHER Organic material Unidentified animal parts

77 Table 9. Percent volume and frequency of prey items consumed by Cyclothone pseudopallida collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 12 n = 90 n = 95 n = 34 n = 46 n = 34 n = 21 E = 11 E = 71 E = 70 E = 25 E = 37 E = 22 E = 20 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F COPEPODA Aegisthus mucronatus Aetideidae Calanoida Chiridus sp Copepoda Harpacticoida Lubbockia aculeata Lubbockia squillimana Lubbockia sp Lucicutia sp Mormonilla phasma Oithona sp Rhincalanus sp Pleuromamma xiphias Pleuromamma sp Valdiviella minor Unidentified copepod parts CRUSTACEA Unidentified crustacean parts

78 Table 9 cont. OSTRACODA Conchoecinae Halocyprididae Myodocopida Ostracoda Unidentified ostracod parts OTHER < Animalia Nematoda < Organic material < Unidentified animal parts

79 Table 10. Percent volume and frequency of prey items consumed by Hygophum benoiti collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 6 n = 23 n = 37 n = 10 n = 7 n = 27 n = 1 E = 6 E = 23 E = 37 E = 10 E = 6 E = 11 E = 1 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda COPEPODA Calanoida Candacia curta Candacia pachydactyla Cyclopoida Farranula gracilis Unidentified copepod parts CRUSTACEA Unidentified crustacean parts DECAPODA Decapoda MOLLUSCA Bivalvia OSTRACODA Myodocopida Ostracoda OTHER Organic material

80 Table 11. Percent volume and frequency of prey items consumed by Valenciennellus tripunctulatus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 1 n = 3 n = 37 n = 24 n = 9 n = 44 n = 29 E = 0 E = 0 E = 4 E = 3 E = 4 E = 12 E = 2 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda < Unidentified amphipod parts COPEPODA Aegisthus mucronatus Aetideidae Calanoida Candacia curta Copepoda < Corycaeus sp Cyclopoida Euchaeta sp Harpacticoida Lubbockia aculeata Lubbockia sp Lubbockia squillimana Oithonidae Pleuromamma abdominalis Pleuromamma piseki Pleuromamma robusta Pleuromamma sp

81 Table 11 cont. Pleuromamma xiphias Poecilostomatoida Rhincalanus cornutus Rhincalanus sp Unidentified copepod parts CRUSTACEA Unidentified crustacean parts EUPHAUSIACEA Euphausiidae OSTRACODA Archiconchoecinae Conchoecinae Halocyprididae < Myodocopida Myodocopina Ostracoda < Unidentified ostracod parts OTHER Organic material Nematoda < < Unidentified animal parts <

82 Table 12. Percent volume and frequency of prey items consumed by Diaphus mollis collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 1 n = 0 n = 14 n = 3 n = 4 n = 10 n = 2 E = 0 E = 0 E = 0 E = 2 E = 0 E = 1 E = 0 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F CHAETOGNATHA Sagittoidea Unidentified chaetognath parts COPEPODA Calanoida < Copepoda Cyclopoida Farranula gracilis Pleuromamma sp Unidentified copepod parts CRUSTACEA Unidentified crustacean parts DECAPODA Decapoda < EUPHAUSIACEA Euphausiidae FISH < Unidentified fish parts < MOLLUSCA Gastropoda

83 Table 12 cont. OSTRACODA < Conchoecinae Myodocopida < Ostracoda Unidentified ostracod parts < OTHER < Organic material Nematoda < < Unidentified animal parts < <

84 Table 13. Percent volume and frequency of prey items consumed by Cyclothone pallida collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 18 n = 115 n = 95 n = 32 n = 58 n = 40 n = 4 E = 15 E = 109 E = 84 E = 32 E = 58 E = 39 E = 4 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Unidentified amphipod parts COPEPODA Aetideidae Haloptilus oxycephalus Unidentified copepod parts CRUSTACEA Unidentified crustacean parts OSTRACODA Conchoecinae Halocyprididae Myodocopida Unidentified ostracod parts OTHER Organic material

85 Table 14. Percent volume and frequency of prey items consumed by Vinciguerria poweriae collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 2 n = 6 n = 72 n = 19 n = 7 n = 45 n = 4 E = 0 E = 4 E = 21 E = 14 E = 3 E = 8 E = 2 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Brachyscelus crusculum Brachyscelus sp Eupronoe armata Hyperiidea Primno latreillei Themistella fusca Tryphana malmi Unidentified amphipod parts COPEPODA Calanoida Candaciidae Candacia bipinnata Candacia varicans Copepoda Corycaeus sp Cyclopoida Farranula gracilis Lubbockia sp. < Paracandacia bispinosa Paracandacia simplex

86 Table 14 cont. Pleuromamma sp Sapphirina sp Temora sp. < Undinula vulgaris Unidentified copepod parts CRUSTACEA Unidentified crustacean parts DECAPODA Decapoda Unidentified decapod parts EUPHAUSIACEA Euphausiidae FISH Myctophidae Unidentified fish parts MOLLUSCA Gastropoda OSTRACODA Archiconchoecinae Conchoecinae Halocyprididae Halocypridinae Halocypris sp Myodocopida Myodocopina Sarsiellidae Unidentified ostracod parts

87 Table 14 cont. OTHER Organic material Unidentified animal parts < <

88 Table 15. Percent volume and frequency of prey items consumed by Myctophum affine collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 1 n = 10 n = 40 n = 6 n = 0 n = 4 n = 0 E = 0 E = 9 E = 21 E = 2 E = 0 E = 0 E = 0 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda Hyperiidea Paratyphis sp Unidentified amphipod parts CHAETOGNATHA Unidentified chaetognath parts COPEPODA Calanoida Candaciidae Copepoda Corycaeidae < Cyclopoida Farranula gracilis Microsetella rosea Oncaeidae Temora sp Unidentified copepod parts CRUSTACEA Unidentified crustacean parts

89 Table 15 cont. DECAPODA Decapoda MOLLUSCA Gastropoda OSTRACODA Conchoecinae Halocyprididae < Myodocopida Ostracoda < OTHER Animalia < Organic material Phytoplankton Unidentified animal parts <

90 Table 16. Percent volume and frequency of prey items consumed by Argyropelecus aculeatus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 3 n = 0 n = 22 n = 0 n = 4 n = 8 n = 3 E = 0 E = 0 E = 7 E = 0 E = 1 E = 2 E = 0 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda Anchylomera blossevillei Eusiridae Gammaridea Hyperiidea Phronima sp Primno evansi Primno sp Scina oedicarpus Unidentified amphipod parts CHAETOGNATHA Sagittoidea COPEPODA Aetideus acutus Calanoida Copepoda Corycaeus sp Cyclopoida Lubbockia aculeata Paracalanidae

91 Table 16 cont. Paracalanus aculeatus Pleuromamma robusta Pleuromamma xiphias Poecilostomatoida Unidentified copepod parts CRUSTACEA Crustacea Unidentified crustacean parts DECAPODA Unidentified decapod parts EUPHAUSIACEA Euphausiidae MOLLUSCA Gastropoda Unidentified cephalopod parts OSTRACODA Archiconchoecinae Conchoecinae Halocypridinae Halocypris sp Myodocopida Ostracoda Unidentified ostracod parts OTHER Organic material Unidentified animal parts

92 Table 17. Percent volume and frequency of prey items consumed by Argyropelecus hemigymnus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 1 n = 2 n = 8 n = 7 n = 3 n = 6 n = 3 E = 1 E = 0 E = 5 E = 4 E = 1 E = 5 E = 3 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Unidentified amphipod parts COPEPODA Calanoida Copepoda Lubbockia aculeata Lubbockia sp Pleuromamma abdominalis Unidentified copepod parts CRUSTACEA Crustacea Unidentified crustacean parts EUPHAUSIACEA Nematoscelis microps OSTRACODA Conchoecinae Myodocopida Ostracoda Unidentified ostracod parts OTHER Organic material

93 Table 18. Percent volume and frequency of prey items consumed by Pollichthys mauli collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 0 n = 0 n = 2 n = 0 n = 15 n = 21 n = 25 E = 0 E = 0 E = 0 E = 0 E = 6 E = 4 E = 5 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda Unidentified amphipod parts COPEPODA Calanoida Copepoda Corycaeus sp Cyclopoida Harpacticoida Pleuromamma borealis Pleuromamma piseki Pleuromamma sp Unidentified copepod parts CRUSTACEA Unidentified crustacean parts DECAPODA Unidentified decapod parts EUPHAUSIACEA Euphausiidae Nyctiphanes capensis Stylocheiron sp

94 Table 18 cont. Thysanopoda sp OSTRACODA Conchoecinae Halocyprididae Myodocopida Ostracoda Unidentified ostracod parts OTHER Organic material

95 Table 19. Percent volume and frequency of prey items consumed by Benthosema suborbitale collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 13 n = 9 n = 64 n = 7 n = 21 n = 74 n = 46 E = 4 E = 7 E = 33 E = 4 E = 9 E = 15 E = 34 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Anchylomera blossevillei Hyperiidea Platysceloidea Unidentified amphipod parts ANNELIDA Polychaeta CHAETOGNATHA Unidentified chaetognath parts COPEPODA Calanoida Candacia bipinnata Candaciidae Copepoda Corycaeus (Urocorycaeus) furcifer Corycaeus sp Cyclopoida Euchaetidae Harpacticoida < < Labidocera sp Pleuromamma abdominalis

96 Table 19 cont. Pleuromamma borealis Pleuromamma piseki Pleuromamma sp Sapphirina metallina Temora stylifera Unidentified copepod parts CRUSTACEA Crustacea Unidentified crustacean parts DECAPODA Decapoda Unidentified decapod parts EUPHAUSIACEA Euphausiidae FISH Myctophidae OSTRACODA Archiconchoecinae Conchoecinae Halocyprididae Halocypris sp Myodocopida Ostracoda Unidentified ostracod parts OTHER Organic material Unidentified animal parts < <

97 Table 20. Percent volume and frequency of prey items consumed by Lampanyctus alatus collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 6 n = 5 n = 25 n = 12 n = 3 n = 16 n = 5 E = 0 E = 3 E = 4 E = 4 E = 0 E = 4 E = 0 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda Gammaridea Hyperiidea Lestrigonus sp Unidentified amphipod parts COPEPODA Aetideus acutus Calanoida Candacia longimana Copepoda Corycaeus sp Eucalanus sp Oncaea sp Paracandacia simplex Pleuromamma piseki Pleuromamma sp Unidentified copepod parts CRUSTACEA Crustacea

98 Table 20 cont. Unidentified crustacean parts DECAPODA Decapoda EUPHAUSIACEA Euphausiidae Nematoscelis sp FISH < Unidentified fish parts < OSTRACODA Conchoecinae Halocyprididae Myodocopida < Ostracoda Unidentified ostracod parts < SALPIDA Salpidae OTHER Organic material Nematoda

99 Table 21. Percent volume and frequency of prey items consumed by Lepidophanes guentheri collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 15 n = 4 n = 36 n = 7 n = 25 n = 57 n = 13 E = 0 E = 1 E = 11 E = 2 E = 8 E = 9 E = 7 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda Gammaridea Hyperiidea Phronima sp Unidentified amphipod parts COPEPODA < Aetideus acutus Calanoida Candacia curta Candacia sp Copepoda Corycaeus sp Cyclopoida < < Farranula gracilis < Harpacticoida Oncaea sp. Paracandacia simplex Pleuromamma gracilis Pleuromamma piseki Pleuromamma sp

100 Table 21 cont. Unidentified copepod parts CRUSTACEA Crustacea Unidentified crustacean parts DECAPODA Decapoda Unidentified decacod parts EUPHAUSIACEA Euphausiidae Thysanopoda sp FISH < < Unidentified fish parts < < MOLLUSCA Gastropoda OSTRACODA Conchoecinae Halocyprididae Myodocopida Ostracoda Unidentified ostracod parts SALPIDA Salpidae OTHER Organic material Nematoda < <

101 Table 22. Percent volume and frequency of prey items consumed by Notolychnus valdiviae collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 1 n = 14 n = 92 n = 30 n = 71 n = 94 n = 41 E = 0 E = 2 E = 23 E = 13 E = 32 E = 35 E = 21 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F CHAETOGNATHA < Unidentified chaetognath parts < COPEPODA Calanoida < Candaciidae Copepoda Corycaeidae Cyclopoida < < Euchaeta sp Harpacticoida < Lubbockia squillimana < Oncaea sp Paracandacia simplex Pleuromamma gracilis Pleuromamma piseki Pleuromamma sp Pleuromamma xiphias Poecilostomatoida Unidentified copepod parts CRUSTACEA Unidentified crustacean parts

102 Table 22 cont. DECAPODA < < Unidentified decapod parts < < EUPHAUSIACEA Euphausiidae OSTRACODA < Conchoecinae Halocyprididae < Halocypridinae < Myodocopida Myodocopina < Ostracoda Unidentified ostracod parts OTHER Animalia Organic material Nematoda < Unidentified animal parts

103 Table 23. Percent volume and frequency of prey items consumed by Ceratoscopelus warmingii collected from three sites in the Gulf of Mexico (AC601, GC852, AT340) separated by time of day. Night = 2030 to 0530 hr CDT, Day = 0730 to 1830 hr CDT, Twilight = 0530 to 0730 and 1830 to 2030; n = total number of stomachs analyzed; E = number of stomachs empty. AC601 GC852 AT340 Night Day Night Twilight Day Night Twilight n = 5 n = 6 n = 23 n = 5 n = 6 n = 39 n = 4 E = 1 E = 4 E = 4 E = 2 E = 3 E = 1 E = 1 Food Item %V %F %V %F %V %F %V %F %V %F %V %F %V %F AMPHIPODA Amphipoda Hyperiidea Phronima stebbingii Unidentified amphipod parts ANNELIDA Unidentified polychaete parts CHAETOGNATHA Unidentified chaetognath parts CNIDARIA Hydrozoa COPEPODA Calanoida Candacia sp Copepoda Corycaeidae Corycaeus sp Cyclopoida Harpacticoida Microsetella rosea Miracia efferata

104 Table 23 cont. Miraciidae Temora stylifera Unidentified copepod parts CRUSTACEA Crustacea Unidentified crustacean parts DECAPODA Caridea Decapoda Unidentified decapod parts EUPHAUSIACEA Euphausiidae FISH Unidentified fish parts MOLLUSCA Bivalvia Cephalopoda Gastropoda Mollusca OSTRACODA Conchoecinae Halocyprididae Myodocopida Ostracoda Platycopida Unidentified ostracod parts SALPIDA Salpida

105 Table 23 cont. OTHER Organic material Unidentified animal parts

106 Table 24. Mean (± 1 standard error) δ 13 C and δ 15 N values for midwater fishes, invertebrates and carbon sources collected from each site (AC601, AT340, GC852). n = number in parentheses, * = multiple fish species grouped together δ 15 N δ 13 C Species AC601 GC852 AT340 AC601 GC852 AT340 FISH Gonostomatidae Cyclothone acclinidens 9.52 (1) (1) Cyclothone alba 7.32 ± 0.32 (5) ± 0.12 (5) Cyclothone pallida 8.44 ± 0.28 (5) 9.97 (1) 8.56 ± 0.63 (4) ± 0.12 (5) (1) ± 0.15 (4) Cyclothone pseudopallida 7.61 ± 0.17 (4) 7.82 ± 0.21 (3) 7.64 ± 0.28 (3) ± 0.41 (4) ± 0.29 (3) ± 0.35 (3) Gonostoma elongatum 8.96 ± 0.27 (6) 7.22 ± 0.23 (6) ± 0.31 (6) ± 0.11 (6) Sternoptychidae Argyropelecus aculeatus 7.90 ± 0.44 (5) 8.68 ± 0.24 (5) ± 0.10 (5) ± 0.41 (5) Argyropelecus hemigymnus 7.82 (1) 7.80 ± 0.22 (9) (1) ± 0.18 (9) Sternoptyx spp.* 8.60 ± 0.26 (7) 6.99 ± 0.89 (2) ± 0.12 (7) ± 0.16 (2) Valenciennellus tripunctulatus 9.15 ± 0.09 (5) 9.06 ± 0.21 (5) 8.57 ± 0.28 (5) ± 0.14 (5) ± 0.16 (5) ± 0.41 (5) Phosichthyidae Pollichthys mauli 6.60 ± 0.19 (10) ± 0.10 (10) Vinciguerria poweriae 7.94 ± 0.10 (10) 7.59 ± 0.32 (5) ± 0.09 (10) ± 0.09 (5) Stomiidae* 8.37 ± 0.26 (11) 7.90 ± 0.95 (6) ± 0.32 (11) ± 0.37 (6) Chauliodus sloani 8.03 ± 0.24 (5) 9.11 (1) ± 0.54 (5) (1) Myctophidae Benthosema suborbitale 6.80 ± 0.42 (3) 7.26 ± 0.49 (3) ± 0.30 (3) ± 0.19 (3) Ceratoscopelus warmingii 6.36 ± 0.29 (3) 7.21 ± 0.33 (5) 6.43 ± 0.63 (4) ± 0.26 (3) ± 0.21 (5) ± 0.35 (4) Diaphus spp.* 8.41 (1) 8.76 ± 0.71 (5) (1) ± 0.28 (5) Diaphus problematicus 9.20 (1) 7.97 ± 0.26 (6) (1) ± 0.08 (6) Lampanyctus alatus 7.91 ± 0.22 (3) 8.48 ± 0.19 (7) 7.49 ± 0.29 (5) ± 0.22 (3) ± 0.13 (7) ± 0.19 (5) Lepidophanes guentheri 7.94 ± 0.25 (4) 6.75 ± 0.23 (6) ± 0.10 (4) ± 0.04 (6) Myctophum affine 5.87 ± 0.28 (10) ± 0.22 (10) 95

107 Table 24 cont. Melamphaidae* 8.27 ± 0.21 (5) 8.22 ± 0.63 (4) 8.77 (1) ± 0.07 (5) ± 0.31 (4) (1) CNIDARIA Atollidae 8.52 (1) (1) Atolla vanhoeffeni (1) (1) Rhopalonematidae Colobonema sericeum (1) (1) SALPIDA Salpidae Salpa cylindrica 1.62 ± 0.61 (4) ± 0.22 (4) Salpa sp ± 1.63 (3) 1.08 ± 0.14 (3) ± 0.71 (3) ± 0.43 (3) CEPHALOPODA Bolitaenidae Japetella diaphana 5.67 (1) (1) Enoploteuthidae Ancistrocheirus lesuerii 6.19 (1) (1) Histioteuthidae Stigmatoteuthis arcturi ± 1.04 (3) ± 0.10 (3) GASTROPODA Cavoliniidae Cavolinia tridentata 1.55 (1) (1) Diacavolinia sp (1) (1) (1) (1) CHAETOGNATHA 8.84 ± 1.24 (5) ± 0.19 (5) AMPHIPODA Phrosinidae Anchylomera blossevillei 3.95 (1) (1) Platyscelidae Platyscelidae sp (1) (1) Platyscelus sp ± 0.02 (2) ± 0.10 (2) 96

108 Table 24 cont. Pronoidae Parapronoe sp (1) (1) COPEPODA 3.70 (1) 7.00 ± 1.53 (5) (1) ± 0.32 (5) Megacalanidae Bathycalanus princeps 9.06 ± 0.57 (4) ± 0.34 (4) Pontellidae Labidocera sp ± 0.29 (2) ± 0.18 (2) EUPHAUSIACEA 5.97 ± 0.66 (4) 6.65 ± 0.25 (2) 4.98 ± 0.23 (5) ± 0.26 (4) ± (2) ± 0.24 (5) Euphausiidae Nematodcelis megalops 6.60 ± 0.33 (3) ± 0.46 (3) Thysanopoda sp ± 0.61 (2) ± 0.54 (2) Thysanopoda tricuspida 2.90 ± 0.50 (2) ± 0.41 (2) DECAPODA Benthesicymidae Gennadas valens 6.83 ± 0.12 (4) 7.50 ± 0.32 (6) 6.49 ± 0.10 (4) ± 0.39 (4) ± 0.31 (6) ± 0.14 (4) Oplophoridae Acanthephyra purpurea 7.78 ± 0.63 (4) 6.98 (1) ± 0.14 (4) (1) Systellaspis debilis 6.30 (1) 5.93 ± 0.29 (6) (1) ± 0.07 (6) Sergestidae Sergia sp (1) (1) ZOOPLANKTON 5.96 ± 2.17 (2) 7.43 ± 0.99 (5) 4.34 ± 1.36 (4) ± 0.06 (2) ± 0.70 (5) ± 0.61 (4) AUTOTROPH Sargassaceae Sargassum spp ± 0.33 (3) 1.88 ± 1.00 (5) ± 0.41 (4) ± 0.65 (3) ± 1.05 (5) ± 0.29 (4) Detritus 3.60 ± 2.08 (5) ± 0.92 (5) Phytoplankton 3.82 ± 0.57 (5) 2.36 ± 0.68 (8) 5.25 ± 0.35 (8) ± 0.78 (5) ± 0.83 (8) ± 0.69 (8) 97

109 Table 25. Percent of prey contributions for each midwater fish species using IsoSource. *Results for M. affine are based on the inclusion of chemosynthetic material, as this species was not bound by the photosynthetic based prey sources. Zooplankton Cnidaria Pterapoda Salpida Cephalopoda Family Species 1-99 %ile 1-99 %ile 1-99 %ile 1-99 %ile 1-99 %ile Gonostomatidae Cyclothone alba Cyclothone pallida Cyclothone pseudopallida Gonostoma elongatum Sternoptychidae Argyropelecus aculeatus Argyropelecus hemigymnus Sternoptyx spp Valenciennellus tripunctulatus Phosichthyidae Pollichthys mauli Vinciguerria poweriae Stomiidae Chauliodus sloani Stomiidae Myctophidae Benthosema suborbitale Ceratoscopelus warmingii Diaphus problematicus Lampanyctus alatus Lepidophanes guentheri Myctophum affine* Melamphaidae Melamphaidae

110 Table 25 cont. Decapoda Euphausiid Fish POM Chemo 1-99 %ile 1-99 %ile 1-99 %ile 1-99 %ile 1-99 %ile

111 Table 26. Mean trophic position (TP), one standard deviation (Stdev), range (minimum maximum) and number of fish (n) for each midwater fish species collected in the north-central Gulf of Mexico, using data from stable isotope and gut content analyses. Trophic positions were calculated using modified equations from Vander Zanden et al. (1996) (see methods). GCA SIA Mean TP Stdev Range n Mean TP Stdev Range n Cyclothone alba Cyclothone braueri Cyclothone pallida Cyclothone pseudopallida Gonostoma elongatum Argyropelecus aculeatus Argyropelecus hemigymnus Sternoptyx spp Valenciennellus tripunctulatus Pollichthys mauli Vinciguerria poweriae Chauliodus sloani Stomiidae Benthosema suborbitale Ceratoscopelus warmingii Diaphus problematicus Diaphus spp Hygophum benoiti Lampanyctus alatus Lepidophanes guentheri Myctophum affine Notolychnus valdiviae Melamphaidae

112 Figure 1. Sampling areas in the North-central Gulf of Mexico for midwater fauna, 9-25 August The three cold seep sites (AT340, GC852, AC601) were located on the continental slope at depths > 1000 m. Each dot represents one station. 101

113 Figure 2. Multidimensional scaling (MDS) plot documenting the differences among the gut contents of midwater fishes. Data were based on the Bray-Curtis similarity matrix calculated from standardized, square root transformed, mean volumes of prey (12 general categories). Colors represent the different fish families, red = Gonostomatidae, Orange = Sternoptychidae, Green = Phosichthyidae, Blue = Stomiidae, Purple = Myctophidae. Ca = Cyclothone alba, Cb = Cyclothone braueri, Cp = Cyclothone pallida, Cps = Cyclothone pseudopallida, Ge = Gonostoma elongatum, Aa = Argyropelecus aculeatus, Ah = Argyropelecus hemigymnus, Vt = Valenciennellus tripunctulatus, Pm = Pollichthys mauli, Vp = Vinciguerria poweriae, Cs = Chauliodus sloani, St = Stomiidae, Bs = Benthosema suborbitale, Cw = Ceratoscopelus warmingii, Dm = Diaphus mollis, Hb = Hygophum benoiti, La = Lampanyctus alatus, Lg = Lepidophanes guentheri, Ma = Myctophum affine, Nv = Notolychnus valdiviae. Clusters are defined at 30% (solid black line) and 60% (dashed black line) similarities. 102

114 Figure 3. Relationships among stomach fullness, mean depth of capture and time for midwater fishes. Data were compiled from all sites and excluded specimens that lacked depth data. A) C. alba, B) C. braueri, C) C. pseudopallida, D) C. pallida, E) A. hemigymnus, F) V. tripunctulatus, G) G. elongatum, H) A. aculeatus, I) P. mauli, J) V. poweriae, K) B. suborbitale, L) C. warmingii, M) D. mollis, N) H. benoiti, O) L. alatus, P) L. guentheri, Q) N. valdiviae, R) M. affine, S) C. sloani, T) Stomiidae. 103

115 A Time 00:00 04:00 08:00 12:00 16:00 20:00 00: B Mean Depth (m) Mean Depth (m) Cyclothone alba (n = 231) 00:00 04:00 08:00 12:00 16:00 20:00 00: C 1300 Cyclothone braueri (n = 233) 00:00 04:00 08:00 12:00 16:00 20:00 00: Mean Depth (m) Cyclothone pseudopallida (n = 310) 104

116 D Time 00:00 04:00 08:00 12:00 16:00 20:00 00: E Mean Depth (m) Mean Depth (m) Cyclothone pallida (n = 328) 00:00 04:00 08:00 12:00 16:00 20:00 00: F 1300 Argyropelecus hemigymnus (n = 14) 00:00 04:00 08:00 12:00 16:00 20:00 00: Mean Depth (m) Valenciennellus tripunctulatus (n = 135) 105

117 G Time 00:00 04:00 08:00 12:00 16:00 20:00 00: H Mean Depth (m) Mean Depth (m) Gonostoma elongatum (n = 78) 00:00 04:00 08:00 12:00 16:00 20:00 00: I 1300 Argyropelecus aculeatus (n = 29) 00:00 04:00 08:00 12:00 16:00 20:00 00: Mean Depth (m) Pollichthys mauli (n = 58) 106

118 J Time 00:00 04:00 08:00 12:00 16:00 20:00 00: K Mean Depth (m) Mean Depth (m) :00 04:00 08:00 12:00 16:00 20:00 00: Vinciguerria poweriae (n = 139) 1300 Benthosema suborbitale (n = 193) L :00 04:00 08:00 12:00 16:00 20:00 00: Mean Depth (m) Ceratoscopelus warmingii (n = 75) 107

119 M Time 00:00 04:00 08:00 12:00 16:00 20:00 00: N Mean Depth (m) Mean Depth (m) Diaphus mollis (n = 30) 00:00 04:00 08:00 12:00 16:00 20:00 00: O 1300 Hygophum benoiti (n = 104) 00:00 04:00 08:00 12:00 16:00 20:00 00: Mean Depth (m) Lampanyctus alatus (n = 63) 108

120 P Time 00:00 04:00 08:00 12:00 16:00 20:00 00: Q Mean Depth (m) Mean Depth (m) Lepidophanes guentheri (n = 127) 00:00 04:00 08:00 12:00 16:00 20:00 00: R 1300 Notolychnus valdiviae (n = 312) 00:00 04:00 08:00 12:00 16:00 20:00 00: Mean Depth (m) Myctophum affine (n = 40) 109