Trophic dynamics and organic carbon sources in Washington DC, USA area seepage spring communities

Seepage springs are a common freshwater habitat in the Washington D.C. area that are home to a variety of invertebrates including the endemic and rare Stygobromus hayi, the official amphipod of Washington DC, USA, but little is known about the community structure of these habitats. We analyzed the general food web dynamics of two seepage springs (Pimmit Run and Goldmine Tract) in the Washington D.C. area that are known to include a congener of S. hayi, Stygobromus tenuis potomacus. Within these two seepage springs, we identified 29 taxa, with varying degrees of abundances, from 6 field excursions over three seasons: Winter, Spring, and Summer. Using dual abundance stable isotope analysis of δ13C and δ15N, we discerned the trophic positions of Lumbriculidae, Conasellus kenki, S. tenuis potomacus, and Crangonyx shoemakeri, and estimated the trophic positions of Tipula, Pseudolimnophila, and a species of Platyhelminthes. S. tenuis potomacus was the dominant predator in both locations with δ15N and δ13C values between 7 and 10‰ and -25 and -27‰ respectively. C. kenki and C. shoemakeri were consistently lower in the food web, with δ15N and δ13C values between 4 and 7‰ and -24 and -27‰ respectively and, in some cases, C. kenki maybe prey for S. tenuis potomacus. In both seeps leaf material was 4 to 5‰ lower in δ13C than the shredding/grazing invertebrates suggesting that they derive nutrients from microbes colonizing leaves not from leaf carbon itself. Trophic dynamics and organic carbon sources in Washington DC, USA area seepage spring communities William Farmer 1, Dan Fong 1 11† Deceased. Date of Passing: April 25, 2025. and Stephen E. MacAvoy 2 1. Department of Biology, American University, Washington DC, USA 2. Department of Environmental Science, American University, Washington DC, USA

Seepage springs are a common freshwater habitat in the Washington D.C. area that are home to a variety of invertebrates including the endemic and rare Stygobromus hayi, the official amphipod of Washington DC, USA, but little is known about the community structure of these habitats. We analyzed the general food web dynamics of two seepage springs (Pimmit Run and Goldmine Tract) in the Washington D.C. area that are known to include a congener of S. hayi, Stygobromus tenuis potomacus . Within these two seepage springs, we identified 29 taxa, with varying degrees of abundances, from 18 field excursions over three seasons: Winter, Spring, and Summer. Using dual abundance stable isotope analysis of δ 13 C and δ 15 N, we discerned the trophic positions of Lumbriculidae , Conasellus kenki, S. tenuis potomacus, and Crangonyx shoemakeri, and estimated the trophic positions of Tipula, Pseudolimnophila, and a species of Platyhelminthes . S. tenuis potomacus was the dominant predator in both locations with δ 15 N and δ 13 C values between 7 and 10‰ and -25 and -27‰ respectively. C. kenki and C. shoemakeri were consistently lower in the food web, with δ 15 N and δ 13 C values between 4 and 7‰ and -24 and -27‰ respectively and, in some cases, C. kenki maybe prey for S. tenuis potomacus . In both seeps leaf material was 4 to 5‰ lower in δ 13 C than the shredding/grazing invertebrates suggesting that they derive nutrients from microbes colonizing leaves not from leaf carbon itself.

Seepage Spring, Hypotelminorheic, Stable Isotope Analysis, Stygobromus, Conasellus

The Washington D.C. area contains an abundance of unique freshwater habitats called seepage springs (Pipan et al. 2012, Culver & Pipan, 2014) where groundwater emerges from subterranean hypotelminorheic habitats (Keany et al., 2018). Seepage springs serve as access points to the organisms that live in the hypotelminorheic habitat (Meštrov 1962). The hypotelminorheic was initially described by Meštrov (1962) and later expanded by Culver and Pipan (2014) to be described as a superficial subterranean drainage that is underlain by an aquiclude, typically a clay layer, which later emerges from a slight depression at a seepage window to create a miniature wetland that may dry up seasonally. The crustacean species which inhabit the seepage springs are generally known, although the use of molecular techniques can result in new species or even genera being identified within what was once considered a single taxon. Amphipod and isopod crustaceans are dominant inhabitants of seepage springs in the Mid-Atlantic area of the USA. The local fauna consists of three dominant genera of amphipod crustaceans: Stygobromus, Crangonyx, and Gammarus, and one dominant genus of isopods: Conasellus (described as Caecidotea in Keany et al., 2018). All Stygobromus species are troglomorphic, characterized by their lack of eyes and pigmentation (Culver et al., 2010). The Stygobromus species are within the same family as Crangonyx (Crangonyctidae) and are assumed to have evolved with the hypotelminorheic habitat as their main habitat, leading to these troglomorphic adaptations (Culver et al., 2010). Amphipods in the genus Gammarus are known to typically occupy the seepage spring run, while those in the genus Crangonyx occupy both the hypotelminorheic and the seepage spring run, acting as a stygophile (Culver et al., 2006). Crustacean amphipods and isopods are not the only group of species that inhabit seepage springs. Many different orders of insect larvae, such as Trichoptera, Odonata, and Diptera, as well as Hydrobiid snails in the genus Fontigens, and fingernail clams in the family Sphaeriidae can also be found in these habitats. While they are commonly found in seepage spring environments, whether they are specialists adapted to the hypotelminorheic or generalists occupying the spring run is unclear. While the member species of the seepage communities are well characterized, the trophic ecology of the communities remains understudied and what is known is largely observational. The dominant crustaceans appear to be shredders or grazers or both. They graze on leaves or the bacteria that accumulates on leaves, or a combination thereof. The Stygobromus species are believed to be strictly grazers while Crangonyx, Gammarus, and Conasellus are thought to be omnivorous. Crangonyx, Gammarus, and Conasellus species have both been observed in lab settings to shred leaves and prey on aquatic earthworms of the families Lumbriculidae and Naididae. Crangonyx and Gammarus have also been observed in the lab preying on young Conasellus kenki. Crangonyx species are also known to consume larvae of Aedes mosquitoes and the water flea Daphnia obtusa (Schwartz, 1991). Gammarus minus is known to cannibalize the sick and dying in nutrient deprived environments (Dick, 1995). Although autochthonous production from bacteria and algae (where it occurs) is probably an important source of carbon for the seepage community, allochthonous production, in the form of leaves falling into the seepage springs, is clearly also important (Carroll et al., 2016). The relative importance of these sources, however, remains unknown. Additionally, it is unclear how the flow of energy travels through trophic levels. If the crustaceans are competing for resources, multiple types of organisms would be seen at the same trophic level, occupying the same functional feeding group (Fry, 2006), even if competitive exclusion dictates they would not be consuming the same resource. If there are mainly predator-prey interactions happening in these seepage springs, then unique trophic levels would be indicated by fractionation values of roughly 3-4‰ for δ 15 N and 1-2‰ for δ 13 C (Fry, 2006). Also, allochthonous production and autochthonous production should be isotopically distinct. Allochthonous production will be C3 leaves with a carbon isotope signature generally between -27 and -30‰ while autochthonous production should have a 13 C enriched signal, probably between -20 and -24‰ because there is a lower concentration of CO 2 in water leading to less discrimination against 13 C. Since organisms incorporate the carbon and nitrogen isotope signature of their nutrients, variations in plant carbon and nitrogen isotopes are key to understanding what they are consuming or, more appropriately, what they are not consuming. In several studies, trophic analysis using dual abundance and has effectively answered the question of interaction in spring and cave environments by analyzing autochthonous, chemoautotrophic, or heterotrophic energy flow (Sârbu et al., 1996; Post, 2002; MacAvoy et al 2016). There is some evidence which suggests that Gammarus minus exhibit trophic plasticity, extending beyond their functional feeding group and acting as a scavenger or predator (in caves at least) (MacAvoy et al., 2016). It has also been shown that subterranean amphipods exhibit specialized feeding strategies, indicating that competition is driving niche partitioning, and that the size of the amphipod does not drive or predict trophic positions (Hutchins et al., 2014). The food webs of seepage communities around Washington DC, USA have not been characterized by stable isotope analysis, and it is important to assess omnivore reliance on animal tissue, and to examine who is eating what within the seepage community. While stable isotopes cannot show, definitively, who is consuming what exactly, they can show what is not consumed and can constrain the possibilities. Stable isotopes could also identify the origin of the organic carbon which fuels the whole system. The objective of this study was to determine the typical food web dynamics and organic carbon sources of two seepage springs. The study also examined the seeps during winter, spring, and summer since the communities may change as water availability fluctuates during the different seasons. STUDY SITES Trophic analysis was performed during each sampling event on the study sites: Pimmit Run Seep C and Goldmine Tract Seep #1 (both in Virginia, USA; discussed in Culver et al. 2023). The goal was to determine the trophic positions of the amphipods present in seepage springs, particularly Stygobromus tenuis potomacus, for whom any information is observational and anecdotal. Of the many seepage springs around Washington DC, these two sites both provide habitat to S. tenuis potomacus, are of similar size, and share many physical characteristics, differing only by their composition of leaf litter and benthic substrate. Identification of the trophic positions of S. tenuis potomacus was deemed important as it is in the same genus as an endangered species endemic to Washington, D.C., Stygobromus hayi, which also lives in seepage spring habitats. COLLECTIONS FOR ISOTOPE ANALYSIS To measure the trophic structure of the seepage springs, carbon and nitrogen stable isotope analysis was performed on each species found at the sites where a representative sample could be obtained. Samples were collected from the seepage springs using a fine-mesh (1mm x 1mm) aquarium net. The net was placed directly downstream of a sediment/detrital area before that area was gently agitated. All debris collected in the net was then transferred to a container and sorted in the laboratory. Sampling at Pimmit Run Seep C occurred during three seasons, Winter (1/16/2017), Spring (5/08/2017), and Summer (9/7/2018). Collections at Goldmine Track Seep #1 were made for the same seasons, Winter (3/7/2018), Spring (6/07/2018), and Summer (9/7/2018). Along with living organisms, wet leaves, dry leaves, soil, and water samples were taken from the seepage springs to characterize the (presumed) base of the trophic system. Between five and ten wet and dry leaves were collected in separate bags and sediment was collected in two 20mL glass vials, one for carbon and one for nitrogen analysis (dual abundance for soil is not possible as removal of carbonates affects the signature of nitrogen). Water from the sites was collected in 500mL NALGENE bottles which were later filtered in the laboratory using glass fiber filters (Whatman 4.7 cm diameter, 1.2 μm pore size) to sterilize the water and to collect any suspended particulate organic matter (SPOM) which may have been present. Glass fiber filters (same type as above) were also deployed at each study site 4-6 weeks prior to sampling to allow bacteria living in the water column to accumulate on the filter. The filters were placed in porous containers directly in the water column with a weight on top to prevent them from being washed out during heavy rains. The weight was not always effective, however, which led to some seasons not having any data for organic matter collected on glass fiber filters. Since most of the organisms in the seepage springs were small (between 1mm and 1cm) and did not provide enough biomass for specialized analysis, the entire organism, or an aggregate of organisms, were used to obtain a signature. Larger organisms like amphipods and isopods that can hold more contents in their digestive systems were placed in clean water with no food for one week, clearing digestive contents. They also underwent lipid extraction via a reflux in dichloromethane for 30 minutes prior to weighing for isotope analysis (Knoff et al. 2002). ISOTOPE ANALYSIS Samples were shipped to the UC Davis stable isotope laboratory where d 13 C and d 15 N were determined using a PDZ Europa ANCA-GSL elemental analyzer coupled to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). The carbon standard was Pee Dee Belemnite, and the laboratory standards were glutamic acid (-11.07 ± 0.05‰, N=11), chitin (-20.45 ± 0.07‰, N=18), Keratin (-24.45 ± 0.04‰, N=8), and Amaranth flour (-12.91 ± 0.08‰, N=9). Quality control and assurance materials have been calibrated against international reference materials, including: IAEA-600, USGS40, USGS41, USGS42, USGS43, USGS61, USGS64, and USGS65. All are directly traceable to the primary isotopic reference material for each element (i.e., VPDB for d 13 C and Air for d 15 N). The computation of the isotope signature was calculated as, δ x E = [( x E / y E ) sample / ( x E / y E ) standard – 1] x 1000 (1) where E is the element in question, X is the atomic weight of the heavy isotope, and Y is the atomic weight of the light isotope (e.g. for carbon, X = 13 and Y = 12). Statistics were completed using JMP 18.0.1. The manuscript text and tables were written using Microsoft Word 16.89.1. Figures were created using Microsoft Excel 16.89.1 and Microsoft PowerPoint 16.89.1.

Trophic analysis using carbon and nitrogen isotopes ratios of the ecosystem components from the two study sites, Pimmit Run Seep C and Goldmine Tract Seep #1, yielded similar results, with the major difference being the leaf and soil signatures (Table 1). Pimmit Run Seep C is dominated by maple leaves whereas Goldmine Tract Seep #1 is dominated by beech leaves. The respective dominant leaf type was analyzed and assumed to be the base of the food web, as it was virtually impossible to obtain usable tissue from the Fontigens snails or Sphaeriidae clams to provide a temporal base (Post, 2002). The signatures of the maple leaf types analyzed at Pimmit Run Seep C are consistent with documented findings of C3 leaves. Our samples do not, however, follow the seasonal variation in δ 13 C ranging from -22‰ to -28‰ in early spring and late fall, respectively, in fresh leaf samples reported by Lowdon and Dyck (1974). This is because leaves that collect within the sweeps during the fall are retained through the spring from the fall season. Seasonal variation is seen, as the both the beech and maple leaf samples became more 13 C enriched as the seasons progressed from spring, to summer, to winter (Table 1). The isotopic signature of the beech leaves from Goldmine Seep had lower d13C and d15N than maple leaves. The data show that the Tipula larvae are within the both the δ 13 C and δ 15 N fractionation ranges (1-2‰ and 3-4‰, respectively) of the soil and leaf litter wherever Tipula were sampled, except for Goldmine Tract Seep #1 in the winter season where it is not within the fractionation range (Figure 2). The aquatic Lumbricidae and terrestrial Lumbriculidae worms are generally within the fractionation range of the soil and leaf samples. In all seasons, except for Goldmine Tract Seep #1 in the winter, the isopod, C. kenki, is within both δ 13 C and δ 15 N fractionation ranges expected for a consumer of Lumbriculidae. In Goldmine Tract Seep #1 in the winter, Lumbriculidae is positioned above C. kenki within the fractionation ranges (Figure 2). When sampled, the Pseudolimnophila and Platyhelminthes were 1-2‰ lower in δ 13 C than C. kenki. For the amphipod, S. tenuis potomacus, the d 15 N ratios are different between the two locations, however that is not the case for d 13 C. In Pimmit Run Seep C, S. tenuis potomacus are more 15 N enriched (9.1 ± 0.8‰ (N32)) than C. kenki ((5.9 ± 0.59‰ (N37)) or any other samples but are slightly less enriched 13 C than C. kenki (-25.4 ± 0.6‰ (N37) and -25.8 ± 0.6‰ (N32) respectively. p=.004). Whereas in Goldmine Tract Seep #1, S. tenuis potomacus are more enriched in both 13 C and 15 N than any other organism and are within the fractionation ranges expected of a consumer of C. kenki. In Pimmit Run Seep C in the winter S. tenuis potomacus is within the fractionation ranges expected of a grazer on organic films of the glass but this was not observed in other seasons or at Goldmine Seep #1. In Goldmine Tract Seep #1 in the summer S. tenuis potomacus was not found, but another amphipod, Crangonyx shoemakeri was found instead. It had a similar isotopic profile as S. tenuis potomacus in Goldmine Tract Seep #1, but slightly less enriched in 15 N. If the S. tenuis is compared to C. kenki from both seeps combined, there isn’t a significant difference in d13C between the two species, however there is for d15N and the difference in means is 3.4‰ (p<0.0001).

The leaf-litter-soil continuum is reflected in the data and follows the increased enrichment patterns related to increased food web depth (Balesdent et al., 1993). Some data show the dry leaf having higher enrichment values than wet leaf (Figures 1 and 2) which is contradictory to the idea that as the leaves decay, they will become more enriched in δ 13 C and δ 15 N. While contradictory, a similar isotope profile has been seen in sandy and waterlogged sites, which are representative of the sites we sampled, with leaf litter being less enriched in δ 13 C than the fall leaves (Balesdent et al., 1993). The lower d13C and d15N in beech relative to maple was because of higher CO2 conductance in beech leaves and nitrogen obtained through fixation (Mariotti, 1983; Fotelli et al., 2003 Unkovich, 2013) (Figure 2). The SPOM naturally falls in the ranges of the leaf-litter-soil continuum due its composition being made up of a combination of the leaf litter and soil. The glass fiber filters were deployed to accumulate bacteria that were in the water column but were often washed out in heavy rains, resulting in only a few data points for them. The glass fiber filters were more enriched than the leaf litter and soil samples in δ 15 N, ranging from 1-5‰, but had roughly the same δ 13 C signature. Earlier work has demonstrated that some organisms in the seep are specialists, namely C. kenki, and F. bottemeri (Pipan et al., 2012). Of these specialists, we report isotope data on seven species: S. tenuis, C. shoemakeri, C. kenki, Platyhelminthes, Lumbriculidae, Pseudolimnophila, and Tipula. Other seep fauna were not analyzed because either the mass needed was not available for the analysis or because they were absent from the seep on the day of collection. Fauna sometimes descend into the soil or burrow in the clay layer because of high temperature or low water availability (Gilbert et al., 2018). Stable isotope analysis was able to discern the trophic positions of Lumbriculidae , Conasellus kenki, Stygobromus tenuis, and Crangonyx shoemakeri, and provided clues to the trophic positions of Tipula, Pseudolimnophila, and Platyhelminthes . It was still unclear exactly what S. tenuis was obtaining its energy from. While stable isotopes cannot definitively say what an organism is consuming, they can show what an organism is not consuming and can constrain the possibilities. It appears that Lumbriculidae worms and Tipula larvae likely occupy the same functional feeding group and are obtaining their energy from biofilms on soil and leaf litter. They are several per mil enriched in 13 C relative to leaves and soils which suggests they are not incorporating leaf or soil carbon directly, but are consuming the nitrogen rich biofilms growing on those substrates. The maple leaf samples did not follow the seasonal variation described previously because the leaves present at the study sites accumulate at once, during the fall season, and are retained through the other seasons. The isopods, C. kenki, are likely obtaining their energy from some combination of the aquatic Lumbriculidae worms and another organism that was not sampled and are assumed to be two trophic positions higher than the base. Stygobromus tenuis potomacus and C. shoemakeri seem to be at different trophic positions, with S. tenuis being higher than C. shoemakeri. C. shoemakeri are likely obtaining their energy from a combination of C. kenki and Lumbriculidae but it is unclear what S. tenuis is obtaining its energy from. It was hypothesized that they could be eating the harpacticoid copepods present in the springs, but an extensive search through 6 liters of leaf litter proved unsuccessful in obtaining enough to get an isotopic profile. The Platyhelminthes and Pseudolimnophila appear as a predator and are likely in the same FFG and trophic position at C. kenki, but our data does not distinguish where they are getting their energy from. The fractionation ranges observed in both locations between leaves and invertebrates suggest that bacterial or fungal films on leaves and soil form the base of the food web with a clear flow of energy to the aquatic Lumbriculidae and Tipula . From there, it appears Lumbriculidae are a potential source of carbon and nitrogen for C. kenki and perhaps C. shoemakeri, although that species was only found at one seep (Goldmine tract seep 1) . It is unclear what could be the major source of carbon and nitrogen for S. tenuis, Pseudolimnophila, and the Platyhelminthes although trophic positions are relatively clear for most organisms sampled. This paper describes the first efforts to identify the community composition and food web dynamics of these understudied seepage spring habitats. While isotopes and not definitively show where an organism gets its carbon and nitrogen, they can exclude sources and constrain the possibilities. More extensive sampling needs to be done to round out the limited view presented here, however the basic outline of the seep’s trophic structure is clear. Acknowledgments. The authors would like to thank the Cave Conservancy of the Virginia’s for their financial support of this work. We also thank Chloe Shostak for her assistance collecting samples and identifying macroinvertebrates.” CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interest.

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Authors Metrics & Citations Metrics Article Usage 156views 81downloads Citations Download citation William Farmer, Dan Fong, Stephen MacAvoy. Trophic dynamics and organic carbon sources in Washington DC, USA area seepage spring communities. Authorea. 28 March 2026. DOI: https://doi.org/10.22541/au.177466178.84049202/v1 DOI: https://doi.org/10.22541/au.177466178.84049202/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.