Pigment-Based Chemotaxonomy of Seagrass Epiphyte Communities; Variables to Consider and uses in Ecosystem Assessment and Monitoring

The report by Orth et al. [1] entitled “A Global Crisis for Seagrass Ecosystems” under the auspices of the Global Seagrass Trajectories Working Group of the National Center for Ecological Analysis and Synthesis, as supported by the National Science Foundation, had the following within its abstract:

“Seagrasses, marine flowering plants, have a long evolutionary history but are now challenged with rapid environmental changes as a result of coastal human population pressures. Seagrasses provide key ecological services, including organic carbon production and export, nutrient cycling, sediment stabilization, enhanced biodiversity, and trophic transfers to adjacent habitats in tropical and temperate regions. They also serve as “coastal canaries,” global biological sentinels of increasing anthropogenic influences in coastal ecosystems, with large-scale losses reported worldwide.”

“The epiphytic algae of seagrasses are important primary producers in seagrass ecosystems and make a significant contribution to food webs”[1]. Numerous studies [2-8] inter alia, including attention in lay literature [9-12] stress the importance of better understanding seagrass/epiphyte ecosystems for a variety of reasons including ecology, tourism, fisheries, sediment stabilization and others.

The 12th International Seagrass Biology Workshop had the theme ‘securing a future for seagrass’ and stated that it was “an important waypoint on the path to greater conservation for seagrass habitats and seagrass-dependent species’ [13]. Seagrasses and their epiphytes have been and are undergoing numerous stressors. Climate change, as pertaining to seagrass ecosystems, includes both global warming resulting from increasing in atmospheric carbon dioxide (CO2) as well as increases in dissolved CO2 and resultant alteration of carbonic acid speciation (i.e., lowered pH=shift to more dissolved CO2/ H2CO3 and lowered ). Additionally, stronger storms and altered precipitation patterns affect seagrass ecosystems. Climate change affects all marine plants and microalgae [14-18]. Nutrient (N,P) pollution, most notably from septic tanks (i.e., onsite sewerage treatment and disposal systems, OSTDS) and agricultural runoff, is well documented to affect seagrass and their epiphytes, as well as extending offshore to alter coral reef ecosystems [19-23].

The importance of the epiphyte biomass within the overall seagrass community cannot be over emphasized. That is, though the seagrass itself is considered the habitat and nursery for a great many species, and obviously food for apex species such as sea turtles and manatees, it is the epiphytes which provide a large part of the base of the food chain for micro- and meso-grazers. These grazers include but are not limited to amphipods, copepods, polychaete worms, molluscs, shrimp and herbivorous fishes [24- 30]. These smaller grazers are then the next part of the food web including many larger fish species [2,6,7, 20].

Seagrass ecosystems are known ‘nurseries’ for a great many species [6,31-33] including the spiny lobster Panulirus argus [34- 36], a target of many sport divers throughout Florida, the Bahamas and the Caribbean. An extremely important economic species in the Bahamas, as it once was in the Florida Keys, is the Queen Conch (Aliger gigas; aka Strombus gigas, Lobatus gigas, Eustrombus gigas). Many studies reveal that newly settled juvenile conch (post-larval stage) prefer seagrass to bare sand [37-39] and that they feed on seagrass epiphytes and detritus [40,41]. The economic impact of the queen conch derives not only from its commercial harvesting as a food source but also from the sport diving industry wherein individuals harvest conch for personal use. Since its popularization by Millie et al. [42], the use of HPLC derived pigment-based chemotaxonomy for the rapid assessment of microalgal communities has become well documented cf [43-61,62].

Marine epiphytes are microalgae and cyanobacteria with associated microbial biomass form the full microbiome that exists on seagrasses and other structures. During the studies we report herein, we emphasized microalgae (diatoms, chlorophytes {green algae}, dinoflagellates, cyanobacteria {aka ‘blue-green algae’}) and cryptophytes, potentially including some macroalgae (i.e., chlorophyte {green algae} and rhodophytes {red algae}), that grow on turtle grass (Thalassia testudinum) in the waters of the Florida Keys, United States and around Eleuthera Island in the Bahamas. Analyses of epiphytes on other seagrasses (Caulerpa prolifera, Halophila wrightii) and macroalgae (Penicillus capitatus, Laurencia sp.) from Florida Bay are also included.

The following quotation is from a chapter by Borowitzka et al. [1] and is presented here in order to emphasize the importance of epiphyte primary production to overall seagrass ecosystem food webs. “The epiphytic algae of seagrasses are important primary producers in seagrass ecosystems and make a significant contribution to food webs. They can account for over 50% of the standing crop in seagrass meadows. In Florida, USA, epiphytic algae contributed [50,62] and 44% of primary production for Syringodium filiforme, Thalassia testudinum, and Halodule wrightii, respectively [63].”

The present paper is meant to emphasize the potential for pigment-based chemotaxonomy in studies of seagrass epiphytes and how these techniques can aid the analyses of the overall ecology of seagrass meadows and adaptive management strategies. Emphasis was placed on determining the best ways to ensure that the entire epiphytic microalgal community is collected and analyzed and how sample collection may alter resultant data. Analyses of seagrass epiphyte communities from a wide variety of sample sites were performed in order to reveal similarities/dissimilarities and potential linkages to nutrient levels, pollution and turbidity.

Fieldwork occurred at several sites within Florida Bay, USA (Figure 1,2) and around the southern part of Eleuthera Island, the Bahamas (Figure 1,3). Seagrass was harvested by hand while free (z<2m) diving. Seagrass blades were cut near their base with scissors and placed into pre-labelled (site, depth, date) large screwtop test tubes in order to capture any epiphytes that may slough off during handling and then are placed in a cooler for transport to the shore-based laboratory of at Florida Atlantic University or the Cape Eleuthera Institute (CEI). Under subdued yellow lighting, Thalassia testudinum or Halodule wrightii had any macroalgal epiphytes Cf [64,65] removed and the blades measured for width (w) and length (l) to determine area (A cm2={2xw}xl). The blades were then gently scraped individually into an aluminum ‘pie plate’ using a polyethylene tissue lifter. The seagrass blades and the tissue lifter were then rinsed with water containing 3.5% salt (NaCl) by weight. In the case of the red algal macrophyte Laurencia, and both the green algae Caulerpa and Penicillus, samples were placed in 3.5% salt (NaCl) water, shaken and sonicated to remove epiphytes. Scrapping the epiphytes off Laurencia, Caulerpa. or Penicillus was obviously impossible due to the shape of their thalli. The water plus non-macroalgal epiphytes was then decanted and filtered through a Whatman 47mm GF/F filter with gentle suction. The filter paper was then folded in half, blotted between paper toweling, folded in half once more (=quartered) and reblotted. The blotted quartered filter was then wrapped in aluminum foil, labelled, and immediately frozen at minus 300C.

Figure 1:Location of Florida Bay, USA and Eleuthera island, the Bahamas.

Copyright©, Pittsburg Post-Gazette, all rights reserved. Reprinted with permission.

Figure 2:Map showing location of Florida Bay at southern tip of Florida and sampling sites within Florida Bay

Figure 3:Map of south Eleuthera with sampling sites indicated by number:

(1) Starve Creek; (2) Poison Point; (3) Patch; (4) Dorm Beach, Site of the Island School & Cape Eleuthera Institute); (5) Paige Creek-In (6) Paige Creek-Out; (7) Sunset Beach; (8) Deep Creek Jetty; (9) Plum Creek; (10) Davis Harbor; (11) Wemyss Bight Beach.

The samples taken at Eleuthera in the Bahamas were stored frozen until the senior author picked them up every 3 months and transported to the laboratory at Florida Atlantic University (FAU) in Boca Raton, Florida. At FAU, each filter was extracted using methanol; acetone; dimethyl-formamide; water (30:30:30:10, v/v/v/v) and analyzed for epiphyte pigments (chlorophylls and carotenoids) in accord with our standard high performance liquid chromatography (HPLC) procedures [32,47,54,66-68]. The FAU (JWL) laboratory uses two Waters 990 and three Waters 996 photodiode array spectrometers gathering HPLC data using Waters 990 or Empower-2 software with Waters Nova Pak 3.9x300 mm C18 columns and Thermo Separation Products P4000 quaternary HPLC pumps. HPLC peak area (AU*min). Data were then entered into an in-house Excel program (‘Pig Calc’) to generate the pigment-based chemotaxonomic assessment of the epiphyte community. Using the integrated peak area values for indicator pigments [Chlorophyll-b(CH Lb)= chlorophytes, fucoxanthin (FUCO)=diatoms, peridinin (PERI)=peridinin-type dinoflagellates, zeaxanthin (ZEA)=cyanobacteria (aka ‘blue-green algae’), and alloxanthin (ALLO)=cryptophytes], the taxon-specific Chlorophyll-a, as proxy for biomass, was calculated to give the Divisional makeup of each epiphyte community. Taxon-specific chlorophyll-a(CH La) concentrations were calculated from pigment data using the following simultaneous linear regression formula as based on molar relationships:

Total CH La=(1.1xZEA)+(2.5xCHLb)+(1.2xFUCO)+(1.5xPERI)+(1.5xALLO)

It must be noted here that the majority of literature reports pigment ratios that are based on weight-to-weight comparisons. It must be noted here that we use molar-to-molar comparisons, as we believe that this better reflects underlying biochemical relationships. An example of a pigment-based chemotaxonomy based on weight relationships of diagnostic pigments is the SLE from Uitz et al. [43] where in the Sum of Diagnostic Pigments

This yields diatoms+dinoflagellates+cryptophytes+chlorophytes+cyanobacteria. We left out the pigments 9-hexanoyloxy-fucoxanthin and 9-butanoyloxyfucoxanthin which were in the full Uitz et al. [43] formula as those pigments is not part of our SLE for epiphytes.

Previous studies on Everglades’ periphyton and epiphytes, between the years 1994-2009 by the senior author, examined various chemotaxonomic mathematical methods to assess these microalgal communities as well as artificial mixtures of single species cultures. These results revealed that the simultaneous linear equation (SLE) and CHEMTAX [55] methods returned quite similar data while the Bayesian Compositional Estimator BCE: [69] performed less well [54,70,71]. Therefore, herein we utilized the SLE equation shown above.

In addition to the analyses of ‘real’ seagrass described above, “epiphytometers” [72,73] aka Artificial Seagrass Units cf. [74-78] were used in our Florida Bay studies. The base is concrete, or sand filled PVC pipe into which 1x20cm deglazed Mylar® strips are inserted and held in place with Marine Goop® adhesive. A small piece of closed cell Styrofoam is glued to the apical end to keep the ‘fake seagrass blade” up in water column and able to bend with current flow, mimicking natural seagrass movement. An example of such an epiphytometer deployed in a Florida Bay seagrass meadow is given here as Figure 4. Epiphyte sampling from the epiphytometer ‘blades’ followed the protocol given above for real seagrass.

Figure 4:Epiphytometer deployed in 1.5m water near Roscoe Key, Florida Bay.

Florida bay studies

Most of the Florida Bay studies took place mainly between 2001and 2003 and many of those results formed the basis for coauthor Singh-White’s Master’s thesis [79]. Reassessment of those data form the present treatise.

Epiphytes on ‘fake’ versus real seagrass blades

It was important to address the potential variability in the chemotaxonomic estimation of epiphyte communities on epiphytometers (fake seagrass) versus that of native seagrass (Thalassia testudinum) collected adjacent to an epiphytometer. The data in Table 1 for ‘real’ seagrass represents data from the epiphytes removed from several blades, whereas the data for the ‘Fake’ (i.e., epiphytometer) samples are for single blades. In this case, the higher cyanobacteria percentage on the real seagrass blades is taken as potentially representing a longer length of time that the real seagrass was present in the field for epiphyte colonization and growth. That is, the “fake” seagrass (i.e., epiphytometer) discussed here was in the water for only 2 months. Within probable error limits, these data reveal quite even epiphyte distributions between real and fake seagrass blades. That is, we found no discrimination of epiphyte taxonomic groups on the epiphytometer ‘blades’ versus native (viz. T. testudinum) seagrass.

Table 1: Epiphytometer (aka fake seagrass) variability data.

Figure 5a is a plot of Chlorophyll-a concentration, as a proxy for epiphyte biomass, versus epiphytometer residence time (0,1,2 months) and shows a comparison of epiphytometer epiphyte concentration/ productivity with that of coincident ‘real’ (i.e., native) seagrass. Figure 5b shows the pigment-based chemotaxonomic assessments of 1-and 2-month epiphytometer samples as compared to native coincident seagrass at Derelict Key. Epiphyte community growth, taken as changes in CHLa concentration, and community Division makeup were found to be easily assessed and monitored using this methodology. Diatoms (Chrysophytes) vastly dominated both epiphytometer and ‘real’ seagrass epiphyte communities (Figure 5b).

Figure 5:(a) Chlorophyll-a concentration on epiphytometer and native seagrass (Thalassia testudinum) blades. (b) Pigment-based chemotaxonomic determined epiphyte divisions on epiphytometer and coincident real seagrass blades.

Potential data variability due to sample site selection

Sample site selection can be also envisioned as potentially biasing results. We sampled around Roscoe Key in central Florida Bay (Figure 2) in order to see how exact site selection (Table 2) may alter resultant data. Table 3 has the micrograms of chlorophyll-a per square centimeter of seagrass blade data as a biomass indicator as well as the pigment-based chemotaxonomic estimation of the epiphyte community. These samples were taken at approximately the same distance (~40-50m) from the key’s shore. The North and West samples were in 0.5m of water whereas the South and East samples were at 1.25m depth. All sites around Roscoe Key were dominated by diatoms with lesser amounts of chlorophytes. The west site also had detectable signals from chlorophytes and cryptophytes as well as having a 3-4-fold higher load of epiphytes.

Given that the year-round prevailing winds in this area are easterly to south-easterly, the higher epiphyte load to the west of Roscoe Key may well reflect a bottom-up control in that primary nutrient (N,P) known to emanate from bird droppings (guano) on Florida Bay keys [80]. That is, based on epiphyte loads on Thalassia leaves and assuming strong bottom-up control, nutrient supply at Roscoe Key appears to be less to the east and higher to the west. The North and South sites are intermediate to the West and East. Future studies such as these should include full primary nutrient analyses (N as total, ammonia, nitrate and nitrate; P as soluble reactive, total and organic).

Potential data variability due to time of year and local conditions

Table 4 contains data collected at the same site within Snake Bight during seven different months. Salinity varied between 24- 4psu. The December 2001 sampling revealed phytoplankton bloom conditions with a total chlorophyll-a concentration of 13.29 mg/L. During this same period, the epiphyte biomass was also greatly enhanced. Together, these values indicate an increased nutrient supply delivered with incoming fresh water as noted by the lowest salinity recorded at this site.

Diatoms dominated during all samplings except for the June 2002 period in which chlorophytes increased significantly. These data support the proposal herein that pigment-based chemotaxonomy is an excellent way to monitor seagrass epiphyte communities. Granted, we do not have the requisite coincident nutrient data for these waters. However, the point of this paper is to reveal the potential for these methods to provide an easy method to assess epiphyte community changes.

Epiphyte load based on sections of the seagrass blade

We compared epiphyte biomass, using chlorophyll-a concentration as a biomass proxy, in relation to the vertical section of Thalassia blades. Top is farthest from, and bottom is close to the seagrass/sediment interface. Middle section is in between the top and bottom sections. A section is defined here as one-third (1/3) of the length of the seagrass blade being sampled.

As can be told from these data, the top of the seagrass blades contains the highest epiphyte biomass. Near Derelict Key, the top of the blades had approximately double the epiphytic biomass compared to the mid and bottom of the blades. Near Roscoe Key, the top section of the blades had 2-3 times as much epiphyte biomass as the mid and bottom sections.

With only minor fluctuations, the taxonomic makeup of these epiphytic communities was essentially equal with diatoms dominating. Figure 5a is the histogram presentation of the epiphyte biomass on the sections of Thalassia blades near Derelict Key. Figure 5b is the graphic representation of the epiphytes on those blades.

Light as a potential bottom-up control of epiphyte biomass

Above (Table 5 and Figure 6), we demonstrated that the epiphyte biomass is highest on the upper one-third of the T. testudinum blades. AS the top of the blades are higher in the water column, the increased productivity/standing crop of epiphytes could be due to higher light levels.

Figure 7 is plot of the photosynthetic active radiation (PAR; 400-700nm) flux versus depth at the Roscoe key site. Within the first meter, PAR decreased by about one-half. Therefore, as photosynthesis requires light, less light will yield less biomass. Over the length (~15-30cm) of a Thalassia blade in Florida Bay the top of the blade will receive more light than the mid or bottom sections. However, it is likely not just the small extinction of light over these short depth changes that control the biomass production noted in the data shown in Figure 6a. Rather, we believe that it is bladeto- blade shading that decreases light levels more than the small (~5-10cm) depth changes over the length of the blade. However, the potential for top-down control of epiphyte biomass on the seagrass blades cannot be overlooked. That is, epiphyte consumers may harvest the lower portion of seagrass blades more easily than the top portion of the blade. Around the clock monitoring of grazer activities, perhaps using cameras such as the well-known GoPro® series, could aid in determining the ultimate cause(s) of lowered epiphyte biomass towards the sediment-water interface as well as aiding the assessment of the overall ecological importance of epiphytes as a primary feedstock.

Figure 6:(a) Epiphyte biomass, using chlorophyll-a concentration as a biomass proxy, in relation to the vertical section of Thalassia blades Derelict key. (b) Pigment-based assessment of epiphyte community structure in relation to the vertical section of Thalassia blades.

Diatoms as major epiphytes on seagrass and macroalgae in Florida Bay

Table 6 contains the pigment-based chemotaxonomic assessment of the epiphytes communities on various seagrass and macroalgae in Florida Bay. This is not meant to be an in-depth survey of the epiphytes on these species but rather just comparative snapshots. As seen from Table 6, diatoms are indeed the vastly major epiphytes on these seagrasses and macroalgae. Dead (brown) Thalassia blades also remain as a substrate for epiphytes.

Table 2: Intra-site variability study. Site locations and depths.

Table 3: Pigment-based chemotaxonomic assessment of epiphytes on T. testudinum from different areas around Roscoe Key in Florida Bay see Table 4.

Table 4: Salinity, Phytoplankton Chlorophyll-a (~biomass) and Thalassia testudinum epiphytes in Snake Bight Florida Bay November 2001 to August 2002.

Table 5: Epiphyte distribution based on section of seagrass (T. testudinum) blades.

Table 6: Epiphytes on various seagrass and macroalgal samples.

Collection and transport of seagrass to ensure analysis of the entire epiphyte community

As given in the Materials and Methods section, seagrass blade plus epiphyte collections were performed by cutting seagrass blades underwater and gently placing them in a pre-labelled screwtop 50mL amber centrifuge tubes. The reason for this is to capture microalgae (epiphytes) that were not strongly attached yet still form part of the overall community.

We performed a verification test on this hypothesis using seagrass collected in December of 2013 from south Savana Sound (25ᵒ02’98” Nx76ᵒ07’28’W) in Eleuthera. Figure 7 contains the divisional estimates of epiphytes scrapped from the Thalassia blades [(Figure 7) bars w. diamonds] and the same sample but including the water in the test tube [(Figure 7) cross hatched bars]. Significant amounts of diatoms and dinoflagellates were found to have been removed from their seagrass communities during the sampling process and/or storage in the test tube before processing in the lab. Pinckney [77] have also noted the sloughing of epiphytes during handling of seagrass. Thus, it is concluded that care during collection, storage and transport must be addressed in order to capture the true total epiphyte community. To ensure that only epiphyte microalgae are being analyzed, one should also analyze the plankton in the water of the seagrass bed. In the present case, the exceeding clear water had very low phytoplankton presence with barely detectable CHLa signals in the 50mL sample bottles (Figure 8).

Figure 7:Light penetration at Roscoe Key south location.

Figure 8:Chemotaxonomic assessment of epiphytes from south Savana Sound seagrass (bars with diamonds= epiphytes scrapped from seagrass blades; cross hatched bars=epiphytes scrapped from seagrass blades plus the water in the collection/transport tube).

Epiphyte community variability in sites around southern Eleuthera

Figure 9 contains two examples of High-Performance Liquid Chromatography (HPLC) separations of pigments extracted from seagrass epiphytes from Eleuthera. Figure 10 contains the derived epiphyte community structures for these two samples.

As can be told from these two cases, as well as the 11 other samples given in Table 7, variable epiphyte communities exist on T. testudinum around Eleuthera Island. One might well expect differences in the primary and secondary consumer communities. Of course, epiphyte yield needs to be examined in ways so as to discern top-down (predation) and bottom up (nutrition/light) controls. It is precisely these differences and the implications derived from such data that the use of pigment-based chemotaxonomy is being suggested herein as a rapid monitoring and adaptive management tool. For example, should a larger quantity of juvenile conch prefer a certain seagrass/epiphyte community, then that could provide background for establishing a Fishery Restricted Area (FRA) for individual cases or a full status Marine Protected Area (MPA), thus benefitting more than one or two specific species. Variations in epiphyte/ grazer communities may well occur through annual cycles and such changes, as noted earlier in text (Table 4) also need to be considered.

Figure 9:HPLC chromatograms of the pigments extracted from epiphytes on seagrass (Thalassia testudinum) recovered from outside Davis Harbor (left) and Starve Creek (right), Eleuthera. Pigment codes: c=Chlorophylls- c1/-c2; P=Peridinin, F=Fucoxanthin, L=Lutein, Z=Zeaxanthin, b=Chlorophyll-b, a=Chlorophyll-a, β=β -carotene, Sr=Scytonemin-reduced, M=Myxoxanthophyll.

Figure 10:Divisional community structure for the epiphytes from seagrass recovered outside Davis Harbor (left) and Starved Creek (right).

Table 7: Epiphytes from Thalassia testudinum sampled at various sites around south Eleuthera Island, the Bahamas.

The examples of pigment-based chemotaxonomically derived seagrass epiphyte communities present herein reveal the utility of this method for rapidly determining community structure. Future studies in which nutrients, grazers, light levels, turbidity and other analyses are included should allow rapid monitoring (weekly, monthly, yearly) of various seagrass epiphyte ecosystems. The use of epiphytometers (aka fake seagrass) also allows a rapid easily monitored method to follow epiphyte productivity. That is, time zero, placement of the epiphytometer, is well known and growth can be assessed on any time scale (day, week, month) the investigator desires.
In Florida Bay, diatoms were most dominant epiphyte group. Around the southern tip of Eleuthera, epiphyte was more variable with diatoms, chlorophytes, cyanobacteria and diatoms forming the community in various percentages. In both Florida Bay and South Eleuthera, epiphyte biomass was observed to be higher near sources of nutrients, mainly in terrestrial runoff. Future studies should obviously include nutrient analyses including nitrogen, phosphorus and iron.
Studies of microalgal senescence and death induced alteration of pigments and pigment ratios [54,66-68,81-84] have shown that various biomarker carotenoids are rapidly altered (e.g., fucoxanthin) or remain unchanged for years (i.e., alloxanthin, lutein, zeaxanthin) during senescence and death. Consideration of the effects of senescence/death [85], sediment resuspension [53] and light levels [47] all enter the proper application of pigmentbased chemotaxonomy [86-105].

Work in Florida Bay was funded by grants to the senior author from the United States Department of Commerce, National Marine Fisheries Division (South Florida Ecosystem Restoration and Management Program) and took place under National Park Service permit numbers Permits # 2000088 and EVER-2001-SCI-0078.

Work around the island of Eleuthera in the in the Bahamas was funded by various research funds obtained from nonrelated contracts to the senior author with the South Florida Water Management District and the National Oceanographic and Atmospheric Administration. Collaborative work with the Cape Eleuthera Institute (CEI) of the Island School and use of their facilities is gratefully acknowledge here. Earth watch Institute volunteers and CEI research assistant Ms. Jessica Rudd working with co-author A. Brooks during collection and processing seagrass at the Eleuthera sites are thanked for their assistances. The Pittsburg Post-Gazette is thanked for permission to use the map shown here as Figure 1.

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