American University Thesis and Dissertation Template for PC 2016

William Farmer

2019

MACROINVERTEBRATE DIVERSITY AND FOOD WEB

DYNAMICS OF SEEPAGE SPRING HABITATS

IN THE WASHINGTON D.C. AREA

William Farmer

ABSTRACT

Seepage springs are a commonly found freshwater habitat in the Washington D.C. area that are home to a variety of invertebrates including Stygobromus hayi, the only endangered species in Washington D.C. and the state amphipod, but little remains about the community structure of these habitats. We analyzed the community structure and general food web dynamics of 4 seepage springs, 1 spring, and 2 streams in the Washington D.C. area. We identified 11975 organisms of 50 unique taxa, 35 insect taxa and 15 non-insect taxa, with varying degrees of abundances from 63 samples over three seasons: Winter, Spring, and Summer. Using Jaccard’s index, it was seen that the community structure of the seepage spring environments in Great

Falls, MD was similar to each other and similar over the three seasons, with an average coefficient of 0.41. While the sites were similar based on presence or absence they were each dominated by different organisms, most being either Lumbriculidae or Caecidotea kenki.

Biodiversity indices of the study sites showed little to no patterns with regard to seasonal shifts or amphipod specificity. Using dual abundance stable isotope analysis of δ13C and δ15N the trophic positions of Lumbriculidae, C. kenki, Stygobromus tenuis, and Crangonyx shoemakeri were discerned, with estimates to the trophic positions of Tipula, Pseudolimnophila, and

Platyhelminthes. It is still unclear exactly what S. tenuis was obtaining its energy from.

ACKNOWLEDGMENTS

I would like to thank the entire Department of Biology at American University for their personal and professional support in the completion of this project and my education, and for their enthusiasm to engage students, like myself, and promote scientific research. I would like to thank my committee members for their consistent input and help, without which this project would not be of the caliber that it is. Most importantly, I would like to thank Dr. Fong for his undying support, not only for the completion of this project, but also for assisting me, and helping guide me, in my pursuit of education and professional development. His guidance and mentorship are something that I am forever grateful for, and I am very happy to have him as a mentor and a friend.

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TABLE OF CONTENTS ABSTRACT ...... ii

ACKNOWLEDGMENTS ...... iii

LIST OF TABLES ...... v

LIST OF ILLUSTRATIONS ...... vi

INTRODUCTION ...... 1

MATERIALS AND METHODS ...... 5

Aim 1 & 2: Community Composition and Comparisons...... 5

Study Sites ...... 5 Physicochemical Measurements ...... 10 Collections and Processing ...... 11 Aim 1: Community Composition...... 11 Aim 2: Community Comparisons ...... 12

Aim 3: Food Web Dynamics ...... 13

Study Sites ...... 13 Collections for Isotope Analysis ...... 14 Computation of Isotope Ratio ...... 15

RESULTS ...... 16

Seep Parameters ...... 16 Community Composition and Biodiversity Estimates ...... 19 Community Comparisons ...... 30 Trophic Analysis ...... 33 Discussion ...... 36

APPENDIX A ...... 41

REFERENCES ...... 55

LIST OF TABLES

Table 1: Study Site Characteristics...... 17

Table 2: Physicochemical Parameters...... 18

Table 3: Descriptive Statistics...... 23

Table 4: Heat Map of Location...... 25

Table 5: Unique Taxa for Each Study Site...... 26

Table 6: Similarity Coefficients Between Seasons...... 31

Table 7: Threshold Similarity Values...... 31

Table 8: Inter-Site Similarity Coefficients...... 32

Table 9: Biodiversity Estimates...... 41

Table 10: Similarity Coefficients in Winter...... 42

Table 11: Similarity Coefficients in Spring...... 43

Table 12: Similarity Coefficients in Summer...... 44

Table 13: Raw Isotope Values ...... 48

LIST OF ILLUSTRATIONS

Figure 1: The General Locations of the Study Sites Relative to Washington D.C...... 7

Figure 2: The Map Above Shows the General Location of the Seepage Spring Named Pimmit Run...... 8

Figure 3: The Map Above Shows the General Locations of the Seepage Springs Located Within Great Falls Park...... 9

Figure 4: An Altered Map from Figure 3 Highlighting Each Study Site Found in the Great Falls, MD Location...... 10

Figure 5: Pie Charts Containing the Three Most Dominant Taxa Plus Everything Else for Each Site Totaled Across All Seasons...... 24

Figure 6: Rank-Abundance Curves for All Study Sites and All Seasons...... 27

Figure 7: Bar Charts of Simpson’s Diversity Coefficients from the Jackknifed Samples...... 28

Figure 8: Bar Charts of Shannon’s Diversity Coefficients from the Jackknifed Samples...... 29

Figure 9: Dual Abundance Isotope Signatures in Pimmit Run Seep C...... 35

Figure 10: Dual Abundance Isotope Signatures in Lower Seep...... 36

Figure 11: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Winter Season in Pimmit Run Seep C...... 45

Figure 12: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Spring Season in Pimmit Run Seep C...... 45

Figure 13: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Summer Season in Pimmit Run Seep C...... 46

Figure 14: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Winter Season in Lower Seep...... 46

Figure 15: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Spring Season in Lower Seep...... 47

Figure 16: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Spring Season in Lower Seep...... 47

INTRODUCTION

The Washington D.C. area contains a plethora 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 at a slight depression or seepage window to create a miniature wetland. The discharge of groundwater at these sites may be severely curtailed or even cease to flow, especially during dry conditions and during summers with a high rate of evapotranspiration of groundwater from the soil to the atmosphere by plants (Culver & Pipan,

2009). The seepage springs are characterized by the presence of blackened, decaying leaves and subterranean fauna (Culver & Pipan, 2014). Some of the organisms are absent from seepage springs when the discharge is severely curtailed or has ceased, and it is usually assumed that they have retreated to the subterranean hypotelminorheic habitat (Gilbert et al., 2018).

Amphipod and isopod crustaceans are common inhabitants of seepage springs in the

Washington, D.C., area. The fauna consists of three dominant genera of amphipod crustaceans:

Stygobromus, Crangonyx, and Gammarus, and one dominant genus of isopods: Caecidotea

(Keany et al., 2018). All Stygobromus species are troglomorphic, characterized by their lack of eyes and pigmentation (Culver et al., 2010) and are assumed to have evolved with the hypotelminorheic habitat is 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, meaning they use the subterranean habitats in part

(Culver et al., 2006).

These seepage spring environments are ecotones, where surface species and groundwater species co-occur, by serving as a border between two habitats (Décamps & Naiman, 1990). The seepage window, or discharge point, acts as the ecotone, bordering the hypotelminorheic habitat and the seepage spring run habitat. The groundwater species, such as Stygobromus and

Crangonyx, inhabit the underground hypotelminorheic environment with varying levels of dependence on the seepage spring environment (Culver et al., 2006), and are found mainly near the seepage window and rarely in the seepage spring run only a short distance from the seepage window. The surface organisms, such as Gammarus and Caecidotea are likely adapted to occupy the seepage spring run habitats.

The amphipod and isopod crustaceans are not the only taxa occurring in seepage springs.

Larvae of many different orders of insects, such as Trichoptera, Odonata, and Diptera, as well as hydrobiid snails in the genus Fontigens, fingernail clams in the family Sphaeriidae, and aquatic oligochaetes also occur in these habitats. Whether or not they are specialists adapted to the hypotelminorheic or generalists occupying the spring run is unclear. The physicochemical parameters of many seepage springs in Washington D.C. have been analyzed (Keany et al.,

2018), while they are good predictors of the presence of crustacean species, they are not good predictors of the presence of any specific species (Keany et al., 2018).

Each of these commonly found crustaceans are generally assumed, to be shredders or grazers or both. They shred and consume the leaf-litter or graze on the bacteria and fungi that accumulate on the leaf-litter. The Stygobromus species are believed to be strictly grazers while

Crangonyx, Gammarus, and Caecidotea are more omnivorous. Crangonyx, Gammarus, and

Caecidotea 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 Caecidotea 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 on the sick and dying in nutrient deprived environments (Dick, 1995). While it is generally known what the crustaceans eat, aside from

Stygobromus, the diet of surface organisms in the seepage springs is undocumented. It is also unclear if the groundwater and surface organisms are interacting at the ecotone; through competition for resources or by predator-prey interactions.

Trophic analysis using dual abundance δ13C and δ15N has effectively answered the question of interaction in multiple cases, in spring and cave environments, by analyzing autochthonous, chemoautotrophic, or heterotrophic energy flow (Sârbu et al., 1996; Post, 2002;

Carrol et al., 2016; MacAvoy et al 2016). It has been shown that Gammarus minus exhibit trophic plasticity, extending beyond their functional feeding group and acting omnivorously

(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) Examining the variation in population characteristics and species abundance over time is important as a changing abundance of organisms can affect the trophic positions. Due to these habitats being ephemeral, as well as because local populations can co-exist and recolonize (i.e., metapopulations [Levins, 1969]), study sites cannot be expected to be 100% similar despite being in close proximity to one another. If one trophic model is used and assumed to be true,

there would be a failure to incorporate any seasonal changes in species presence or abundance that may impact the seepage spring habitat.

For this study, we aim to determine the following:

1. The macro-invertebrate community composition.

2. The extent of variation of macro-invertebrate community structure over time and space.

3. Community interactions at the ecotone; food web dynamics of these seepage springs.

MATERIALS AND METHODS

Aim 1 & 2: Community Composition and Comparisons

Random quantitative sampling near the seepage window can address questions 1 and 2.

Biodiversity indices and counts of species richness produced from random sampling have been used to compare seepage spring habitats and spring habitats in New Zealand where it was seen that seepage spring environments had a large proportion of Diptera taxa, and a large abundance of mollusks present (Hawksworth & Bull, 2006), but nothing has been done in the Washington

D.C. area. Keany et al. (2018) identified crustaceans in seepage springs but did not identify insects present. Morris et al. (2014) showed that the use of specific biodiversity indices can produce vastly different results and recommends using more than one index simultaneously, and that some indices are not fit for non-random samples. Identifying if the same species are present between sites and seasons can be done by using various similarity coefficients, each with different implications (Chao et al., 2006).

Study Sites

Five study sites are chosen based on their proximal location to one another, ease of access, and the inhabitance of an abundance of amphipod and isopod species. Four of the study sites are seepage spring habitats and the fifth is a permanent spring habitat. Three seepage springs, North Seep, Upper Seep and Lower Seep as well as the permanent spring, Valley Trail

Spring, are located within Great Falls Park, Maryland. Water from North Seep flows into the

C&O Canal while water from the other sites flow into the Valley Trail Stream before emptying into the C&O Canal. The fourth seepage spring, Pimmit Run Seep C, is situated in the George

Washington Memorial Parkway near Chain Bridge in Virginia. Water from Pimmit Run Seep C

flows into Pimmit Run before emptying into the Potomac River. The locations of these sites are given in Figures 1 - 4. The foliage dominating Pimmit Run Seep C is comprised of maple and beech while oak and beech dominate the other study sites. All the study sites have adequate tree coverage and the seepage spring surfaces are covered in leaves throughout the entire year. These study sites and their baseline main streams (Valley Trail Stream and Pimmit Run) were sampled in the winter, spring, and summer seasons. Sampling included measurements of physical and chemical parameters, collections for biodiversity estimates, and collections for trophic analysis.

Figure 1: The General Locations of the Study Sites Relative to Washington D.C. Washington D.C. is outlined as the beige diamond-like area, with American University located at the flag. The baseline main streams are indicated by the text and arrow, and the number in parenthesis identifies how many study sites are at each location. Pimmit Run is in Virginia, just across the Potomac river, and contains Pimmit Run Seep C and Pimmit Run Main Stream. The other study sites: North Seep, Upper Seep, Lower Seep, Valley Trail Spring, and Valley Trail Main Stream are located within Great Falls Park in Maryland. Figure 2 details the location of the sites at Pimmit Run and Figures 3-4 detail the locations of the sites within Great Falls, MD.

Figure 2: The Map Above Shows the General Location of the Seepage Spring Named Pimmit Run. Pimmit Run Seep C is indicated by the dot, which eventually flows into the Pimmit Run Main Stream, which then flows into the Potomac River.

Figure 3: The Map Above Shows the General Locations of the Seepage Springs Located Within Great Falls Park. Access to the park is from the Anglers entry point, and the sites are accessed just off the trails. The area outlined in the red square is expanded on in Figure 4.

Figure 4: An Altered Map from Figure 3 Highlighting Each Study Site Found in the Great Falls, MD Location. Legend and scale bars are provided. North seep flows into a stream which flows into the C&O canal while Upper Seep flows into the Valley Trail Main Stream first, followed by Valley Trail Spring and Lower Seep. Samples taken from Valley Trail Main Stream were just above the discharge point of Valley Trail Spring.

Physicochemical Measurements

At each site at each sampling season a YSI Pro Plus multi-meter will be used to obtain the following: Barometric pressure (mmHg), Temperature (oC), Dissolved Oxygen (%, and mg/L), Specific Conductivity (SPC, μS/cm), Conductivity (μS/cm), Total Dissolved Solids

(TDS, mg/L), and pH.

Collections and Processing

To determine community composition and structure, three samples of the fauna were taken at each site at each sampling season as close to the seepage window as possible. Each sample was collected by placing a fine-mesh (1mm x 1mm) aquarium fish net in the substrate and disturbing a 15cm x 15cm area immediately upstream which allows the water flow to push the debris through the net. The contents of the net are then placed into a re-sealable plastic bag and transported back to the laboratory in insulated coolers. In the laboratory, live amphipods and isopods were counted, removed, and kept alive for other studies. All other organisms were preserved in 80% ethanol until sorting and counting. All organisms were identified to the lowest taxonomic status possible, without genetic analyses, using various keys.

Aim 1: Community Composition

Diversity was estimated using the Simpson’s index of diversity (D, measured as the inverse of Simpson’s index of dominance) and the Shannon-Wiener index of diversity (H’).

These two indices were chosen because the Simpson’s Index deemphasizes rare species over abundant species while the Shannon-Wiener Index emphasizes rare species over abundant

1 species. Simpson’s index is calculated as, D = 2 , where D is the index, and pi is the fraction ∑(푝푖) of the ith species to the entire sample. Shannon’s index will be calculated as, H′ = −∑(p푖 ∗ ln(pi)), where H’ is the index and pi is the fraction of the entire sample that is made up by species i. Because we obtained three samples at each collection event, the Jackknifing method was used to obtain the best value of biodiversity (Krebs, 1999). A total of the three samples is obtained and then three jackknifed samples were calculated by summing all possible combinations of two of the three samples. Diversity estimates, such as D or H’, were calculated

from the total sample and each of the jackknifed samples. Each of the jackknifed estimates was converted to a pseudo value as follows: (Dttl * 3) – (Djki * 2), where Dttl is the Simpson’s Index based on the total and Djki is the Simpson’s Index based on Jackknifed sample i. The best estimate of the diversity index is calculated as the average of the three pseudo values. This same concept is applied to Shannon’s index of diversity.

Aim 2: Community Comparisons

To compare the seepage springs to one another Jaccard’s index, Sorenson’s index, Bray-

Curtis index, and percent similarity were used to estimate how similar the biodiversity of the seepage springs are to one another.

a Jaccard’s index is calculated as J = , where J is Jaccard’s coefficient, a is the s a+b+c s number of species found in both sites, b is the number of species found only in site 1, and c is the number of species found only in site 2.

Sorenson’s index is like Jaccard’s, using presence or absence data to establish similarity.

2a It is calculated as S = , where S is Sorenson’s coefficient, a is the number of species s 2a+b+c S found in both sites, b is the number of species found only in site 1, and c is the number of species found only in site 2.

Bray-Curtis index uses abundance data along with presence or absence. The Bray-Curtis similarity index can be calculated two ways, each giving the same result. These calculations are:

2∑min (Xij,Xik) ∑|Xij−Xik| BCs = and BCs = , where BCs is the Bray-Curtis coefficient, Xij is the ∑(Xij+Xik) ∑(Xij+Xik) number of species i in sample j, and Xik is the number of species i in sample k. We used the first method of calculating the Bray-Curtis index.

Percentage similarity was also used in this study to directly compare how similar study sites were based on abundances of organisms present. It was calculated as Ps = ∑min (푝1푖, 푝2푖), where Ps is percent similarity, 푝1푖 is the percentage of species i in site 1, and 푝2푖 is the percentage of species i in site 2.

Aim 3: Food Web Dynamics

Aim 4 can be addressed by using dual abundance stable isotope analysis of δ13C and

δ15N to infer trophic positions. It is known that if competition for resources is happening, multiple types of organisms would be seen at the same trophic level, occupying the same functional feeding group (Fry, 2006). If there are mainly predator-prey interactions happening in these seepage springs, then unique trophic levels would be seen indicated by increased isotope ratios of 3-4‰ δ15N and 1-2‰ δ13C, as this is above the typical fractionation value that is observed when one organism eats another (Fry, 2006). The organisms that are getting their energy mainly from different plant sources have variations in δ13C signature associated with the plants which they are eating (Fry 2006).

Study Sites

Trophic analysis was performed during each sampling event on two study sites: Pimmit

Run Seep C and Lower Seep. The goal was to determine the trophic positions of the amphipods present, specifically Stygobromus tenuis. These two sites were the only sites with S. tenuis and differ by their composition of leaf litter and organisms present. Identification of the trophic positions of S. tenuis was deemed important as it is in the same genus as the only endangered species in Washington, D.C., Stygobromus hayi, which lives in a very similar seepage spring habitat.

Collections for Isotope Analysis

Sample collections from the seepage springs was like that of the biodiversity collections, a fine-mesh (1mm x 1mm) aquarium net is deployed in the seepage spring and a small area of sediment is disturbed directly upstream of the net. All debris collected in the net is then transferred to a container to be taken to and sorted in lab.

Along with living organisms, wet leaves, dry leaves, soil, and water samples were taken from the seepage springs to create a base trophic position. Wet and dry leaves (about 5 to 10) were collected in separate bags while soil 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 effects the signature of nitrogen). Water from the sites was collected in a 500mL NALGENE bottle which was later filtered through a glass fiber filter to sterilize the water and collect any suspended particulate organic matter (SPOM) that is present.

Glass fiber filters 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 deployed in porous containers placed directly in the water column with a weight on top to prevent it from being washed out during heavy rains, which was not always effective

Since most of the organisms in the seepage springs were too small and did not provide enough biomass for specialized analysis (muscle tissue only, etc.), the entire organism, or an aggregate of organisms, were used to obtain a signature. Larger organisms like amphipods and isopods that had their stomach and gut contents cleared by being placed in clean water with no food for one week. This was so that during analysis, we are examining what they have eaten over time, instead of just recently. Some of the smaller organisms that were required to be clustered didn’t undergo isotope analysis since there were not enough to meet the minimum weight requirement (1-2mg), or if it was believed that it would not be a representative sample. 14

Computation of Isotope Ratio

Dual abundance stable isotope analysis of δ13C and δ15N signatures was conducted by

UC Davis. The PDZ Europa ANCA-GSL elemental analyzer coupled to a PDZ Europa 20-20 isotope ratio mass spectrometer was the primary instrument for analysis.

x ( E)sample x yE The computation of the isotope signature was calculated as, δ E = [ x − 1] ( E)standard yE

,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. X = 13 and Y = 12 for carbon).

RESULTS

The study sites were grouped and color coded based on the three dominant amphipods in the seepage spring or stream in all tables and figures, if applicable. North Seep and Upper Seep contained Crangonyx shoemakeri, coded by blue. Lower Seep and Pimmit Run Seep C contained

Stygobromus tenuis, coded by red. Valley Trail Spring and the Valley Trail Main Stream contain

Gammarus minus, coded by green. There were no amphipods found in the Pimmit Run Stream.

This could be due to non-representative sampling at the site as sampling was conducted using a

15cm aquarium fish net, which could not collect a representative sample from the large stream. It was virtually impossible to get to the middle of the stream and collect from the benthic substrate using the same sized net for a quantitative sample which led to a lack of organisms collected from the site. For the remainder of the paper Pimmit Run Stream will be excluded in analysis.

While Lower Seep contained mostly S. tenuis, it was the only study site that contained more than one genus amphipod. At Lower Seep in the winter season, in one non-quantitative sample, G. minus, C. shoemakeri, and S. tenuis were found together. The isopod, Caecidotea kenki, was found in every study site, excluding the Pimmit Run Stream.

Seep Parameters

The seepage springs were all slightly different from one another, according to size, average depth, water flow, leaf litter, and benthic substrate (Table 1). North Seep, Pimmit Run

Seep C, and Valley Trail Spring were the most spring-like in nature, characterized by a moderate flow rate, less leaf coverage, and a non-muddy substrate. Upper Seep and Lower Seep were similar with a slow flow rates, high amount of leaf coverage, and a very muddy substrate.

Due to the various flow rates of the study sites, the water temperatures were variable.

Those with higher flow rates had more consistent water temperatures (Table 2). The amount of

dissolved oxygen also varied among the study sites and it was observed that those with more leaf litter covering the site had less dissolved oxygen in the water (Table 2). Conversely, those with more leaf litter had a higher ppm of dissolved organic carbon (Table 2). The pH of the study sites varied between sites and between seasons but was not found to be associated with any of the other recorded variables. The specific conductivity (SPC) was also not found to be associated or related to any other variables, and its elevation at Pimmit Run Seep C could not be explained

(Table 2).

Table 1: Study Site Characteristics.

Upper Lower Pimmit Run Valley Trail Valley Trail Pimmit Run North Seep Seep Seep Seep C Spring Stream Stream Width, cm 126 46 30 41.6 48.6 84 n.d. Depth, cm 1.62 25.4 1.3 0.83 2.72 4.5 8 Water Flow Moderate Slow Very Slow Moderate Moderate Fast Very Fast Leaf Litter Beech Beech Beech Maple Beech Beech None Percent Covered ~15% >90% >90% ~60-70% ~10% <5% None Substrate Gravel, Sand Mud, Sand Mud, Sand Gravel, Sand Gravel, Sand Gravel, Sand Gravel, Sand Characteristics of the study sites are listed at the location of sampling. Lengths were not measured, as all sites flowed into another stream, and all sampling events were performed at the seepage window, if applicable.

Table 2: Physicochemical Parameters.

North Upper Lower Pimmit Valley Trail Valley Trail Seep Seep Seep Run Seep C Spring Stream Temp, C Winter 11.5 11.8 9.0 12.7 ± 0.2 10.9 7.7 Spring 13.1 ± 1.6 13.2 ± 1.4 12.9 ± 1.8 14.1 ± 1.3 14.3 ± 1.4 16.0 ± 3.2 Summer 18.6 16.4 20.6 17.6 19.0 20.0 DO, mg/L Winter 8.6 7.0 6.5 7.1 ± 0.1 9.0 10.5 Spring 7.7 ± 0.5 5.0 ± 1.5 4.4 ± 1.5 5.9 ± 0.9 8.3 ± 0.3 8.2 ± 0.4 Summer 7.4 2.2 0.2 4.5 5.4 8.2 DO, % Winter 80.3 65.1 57.4 68.8 ± 1.7 83.0 88.9 Spring 74.8 ± 37 49.3 ± 16.7 42.4 ± 15.2 57.3 ± 6.6 82.6 ± 2.3 82.8 ± 3.2 Summer 79.6 20.1 3.0 49.2 57.2 90.6 SPC, 훍S/cm Winter 77.5 190.0 108.8 1530 ± 1.4 145.3 169.7 Spring 81.7 ± 47.3 200 ± 24.2 104.5 ± 9.1 1279 ± 59.4 137.7 ± 7.9 200.2 ± 2.5 Summer 69.3 164.3 126.0 1165.0 121.6 193.4 pH Winter 7.42 6.42 6.23 6.22 ± 0.0 6.63 6.28 Spring 6.52 ± 0.3 6.04 ± 0.1 6.13 ± 0.1 6.17 ± 0.1 6.97 ± 0.1 6.98 ± 0.1 Summer 6.42 6.12 6.05 6.40 6.73 6.90 DOC, ppm Winter 0.5 ± 0.0 1.3 ± 0.0 2.0 ± 0.2 1.8 ± 0.0 0.5 ± 0.0 n.d. Spring 0.6 ± 0.0 0.6 ± 0.0 1.8 ± 0.0 1.2 ± 0.0 0.4 ± 0.0 0.9 ± 0.1 Summer 0.6 ± 0.0 0.7 ± 0.0 2.2 ± 0.0 1.2 ± 0.1 0.5 ± 0.0 n.d. DOC, δ13C Winter -26.0 ± 0.5 -27.4 ± 0.1 -29.5 ± 0.6 -25.1 ± 0.0 -25.9 ± 0.5 n.d. Spring -26.3 ± 0.1 -26.2 ± 0.2 -28.6 ± 0.1 -28.1 ± 0.0 -27.2 ± 0.3 -27.3 ± 0.2 Summer -27.3 ± 0.0 -25.7 ± 0.2 -28.6 ± 0.0 -25.7 ± 0.1 -26.4 ± 0.3 n.d. Sample Sizes* (n) Winter 1 1 1 2 1 1 Spring 3 3 3 2 3 2 Summer 1 1 1 1 1 1 Above shows the physicochemical parameters ± the standard deviation (if applicable) for each study site: Temperature in Celsius, Dissolved Oxygen (DO) in mg/L and % in parenthesis, Specific Conductivity (SPC) in μS/cm, pH, Dissolved Organic Carbon in ppm, and δ13C ratio. The term “n.d.” refers to no data. *Sample sizes for DOC data are 2, 2, 2, except for Pimmit Run Seep C with DOC sample sizes of 4, 2, and 2 for winter, spring, and summer, respectively.

Community Composition and Biodiversity Estimates

For all study sites, 50 unique taxa were observed and identified to the lowest taxonomic status possible. We identified 15 unique non-insect taxa and 35 unique insect taxa. We counted

11975 individuals, 10047 non-insect individuals, and 1928 insect individuals.

Among non-insects, the mollusks (Fontigens bottimeri, Gyro, Ancylidae, and

Sphaeriidae) accounted for 44.9% (4504) of individuals with F. bottimeri accounting for 35.9%

(3603) of individuals. The isopod C. kenki accounted for 24.7% (2481) of all non-insect individuals. All the amphipods combined account for 12.7% (1282) of non-insect individuals with G. minus alone making up 10% (1046) of the total. The oligochaete worms (Lumbriculidae and Naididae) made up for 12% (1207) of non-insect individuals. The rest of the non-insect individuals are made up by Platyhelminthes (4.5%), Nematoda (0.5%), Ostracods (0.5%), and

Copepods (0.2%). The oligochaete worm, Lumbriculidae, was the most commonly found organism, found in 52 of the 63 samples.

All the insects observed were in their aquatic larval stages except for the semi-aquatic

Veliidae and Sminthuridae families. The Diptera order of insects accounted for 67.3% (1297) of all insects counted with the Chironomid family accounting for 60.5% (1168) of all insect individuals counted. The order Plecoptera then made up for 18% (348) of all insect individuals with the Perlidae family accounting for 15% (290) of the insects counted. The Trichoptera order accounted for 10.5% (202) of all insect individuals with the Lepidostomatidae family making up

8.1% (156) of all insect individuals. The rest of the insect individuals are made up by

Ephemeptera (1.6%), Coleoptera (1%), Collembola (0.6%), Odonata (0.4%), Megaloptera

(0.4%), and Hemiptera (0.2%). The Chironomid, Tanypodinae, was the most commonly found insect, found in 45 of 63 samples.

While some of these organisms make up for a large portion of individuals found (e.g.

Fontigens bottimeri making up 35.9%) it is important to know where and what proportion of individuals are found at each site and during what seasons. Table 3 shows a heat map of location that individual taxa are found by displaying the proportion of taxa i based on the sum of taxa i across all sites and seasons. Cells that are shaded green have low abundance while cells shaded red have high abundance. We see from this table that certain organisms are either present only in specific study sites or have a higher presence during specific seasons. These findings are also visualized in Figure 5, which shows a 4-slice pie chart containing the 3 most abundant taxa in each site plus everything else. Gammarus minus is found only in the Valley Trail Spring and

Valley Trail Stream sites, with a higher presence in Valley Trail Spring (Table 4, Figure 5,

E&F). Caecidotea kenki was found in every study site but is more present in Upper Seep, Lower

Seep, and Pimmit Run Seep C (Table 4, Figure 5, B-D). Fontigens bottimeri was found in 3 study sites at each season and 1 study site for 2 seasons but had a high presence at the Valley

Trail Spring site, accounting for 91.4% of all individuals of F. bottimeri found, with an increased presence in the winter season. Platyhelminthes and Lumbriculidae worms were found in each study site with a slightly higher presence in the winter and spring season and made up the top 3 taxa found in 2 and 4 sites, respectively (Table 4, Figure 5). The insects were found to have an even presence across all sites, but being lower in the Pimmit Run Seep C and Valley Trail Spring sites.

To further examine the evenness and richness of the study sites, rank abundance graphs

(Figure 6) were produced displaying the relative abundance of taxa i on the Y axis and the abundance rank of taxa i on the X axis for each taxon found for each study site totaled across all seasons. Ignoring the Pimmit Run Stream, there seems to be no consistent trend based on the

presence or absence of certain amphipods. Additionally, it is observed that the abundance rank curves of each site fit into three patterns: high species richness and evenness, high species richness and low evenness, and low species richness and low evenness. North Seep, Upper Seep,

Lower Seep, and Valley Trail Stream fit to the first pattern. They have higher species richness’s of 29, 27, 24, and 26, respectively with a high evenness, shown by a lower slope (Figure 6, Table

5). Valley Trail Spring fits to the second pattern, with a species richness of 22 but a low species evenness based on the large initial slope of the curve (Figure 6, Table 5). Pimmit Run Seep C fits to the third pattern with a species richness of 14 and a large slope (Figure 6, Table 5). Unique taxa and individual counts used for abundance ranks are displayed in Table 5.

Another way to examine the species richness and evenness is to use biodiversity indices.

We used both Simpson’s Index of Diversity, D, (Inverse of Simpson’s Dominance) and

Shannon’s Index of Diversity, H’. Simpson’s index favors the abundance of species while

Shannon’s index favors species richness and rare species (Krebs, 1999). Results from jackknifing of the 3 samples for each season are displayed in Figure 7, 8 and Supplemental Table 1. It is observed from the biodiversity results of all organisms that there are no major trends regarding seasonal differences in diversity nor are there trends regarding specific amphipod presence.

Some sites showed an increase in diversity from winter to spring then a decrease from spring to summer, while some study sites showed the opposite. Additionally, some sites showed consistent increases in diversity from winter to summer while some showed consistent decreases in diversity. Comparing the sites that have the same amphipod present show no trend, or contradicting trends. Examining the biodiversity results of insects only or non-insects only showed similarly inconsistent results. Ignoring trends between seasons or amphipods, similar patterns displayed in the rank abundance curves are seen (Figure 7, 8). North Seep, Upper Seep,

Lower Seep, and Valley Trail Stream have high diversity indices for both Simpson’s and

Shannon’s Index while Pimmit Run Seep C and Valley Trail Spring have low index values.

Comparing the two indices it is seen that Simpson’s Index of Diversity varies more than

Shannon’s Index of diversity, for all categories (Figure 7, 8). This is likely due to the large amount of species found at low abundance in each study site, which Shannon’s Index of

Diversity favors. Additionally, in each seepage spring there was always a single taxon that made up for at least 25% of the total organisms found (Figure 5, 6; Table 5).

Together, all of these data show that the study sites found in Great Falls, MD follow similar patterns, having a high species richness and relatively high diversity with the exception of Valley Trail Spring as it was dominated by F. bottimeri. The study sites found at Pimmit Run in Virginia had less species richness and less diversity, but since there are not replicates for seepage springs in Virginia it is not appropriate to categorize them as such.

Table 3: Descriptive Statistics.

Pimmit Valley Upper Lower Run Seep Trail Valley Trail Main North Seep Seep Seep C Spring Stream Crangonyx shoemakeri (n) Winter 1 73 0* 0 0 0 Spring 8 48 1 0 0 0 Summer 0 39 2 0 0 0 Stygobromus tenuis (n) Winter 0 0 15 23 0 0 Spring 0 0 2 24 0 0 Summer 0 0 0 9 0 0 Gammarus minus (n) Winter 0 0 0* 0 423 57 Spring 0 0 0 0 382 78 Summer 0 0 0 0 94 12 Caecidotea kenki (n) Winter 47 382 69 424 2 2 Spring 9 243 180 607 1 6 Summer 8 201 40 257 1 2 Total # Individuals (# Unique Taxa) Winter 258 (20) 775 (16) 509 (14) 485 (6) 2554 (17) 151 (16) Spring 589 (23) 602 (17) 885 (20) 708 (10) 1672 (13) 249 (17) Summer 202 (18) 712 (20) 602 (18) 326 (10) 532 (10) 138 (16) Dominant** Organism Caecidotea Caecidotea Fontigens Winter Platyhelminthes kenki Lumbriculidae kenki bottimeri Gammarus minus Caecidotea Caecidotea Fontigens Spring Perlidae kenki Diamesinae kenki bottimeri Gammarus minus Caecidotea Fontigens Summer Lumbriculidae Diamesinae Sphaeriidae kenki bottimeri Lumbriculidae The table above shows general descriptive statistics for each study site for each season: the number of crustaceans for each species found, the total # of individuals and taxa sampled, and the most dominant organism. *In a non-quantitative sample of Lower Seep in the winter season we found G. minus, C. shoemakeri, and S. tenuis. **Dominant organism refers to the most abundant organism in each season in each site.

A North Seep B Upper Seep

Lumbriculidae 279

Caecidotea All else All else 826 403 752

Perlidae 247

Platyhelminthes Lumbriculidae Diamesinae 120 209 302

C Lower Seep D Pimmit Run Seep C Stygobromus All else Sphaeriidae 56 54 547 Platyhelminthes 121 All else 708

Caecidotea 1288

Caecidotea Lumbriculidae 289 452

E Valley Trail Spring F Valley Trail Main Stream All else Lumbriculidae 407 Sphaeriidae 120 172

All else Gammarus 196 899 Fontigens Gammarus 3280 147

Sphaeriidae 75

Figure 5: Pie Charts Containing the Three Most Dominant Taxa Plus Everything Else for Each Site Totaled Across All Seasons

24 Table 4: Heat Map of Location.

Heat Map of North Seep Upper Seep Lower Seep Pimmit Run Seep C Valley Trail Spring Valley Trail Stream location Winter Spring Summer Winter Spring Summer Winter Spring Summer Winter Spring Summer Winter Spring Summer Winter Spring Summer Scale Crangonyx 0.6% 4.9% 39.3% 29.4% 23.9% 0.6% 1.2% 0.0% Stygobromus 30.0% 4.0% 31.5% 48.0% 18.0% 0.1% Gammarus 42.8% 38.6% 9.5% 5.4% 7.9% 1.2% 1.0% Caecidotea 2.3% 0.4% 0.4% 18.6% 11.8% 9.8% 3.4% 8.8% 1.9% 17.1% 29.5% 12.5% 0.1% 0.0% 0.0% 0.1% 0.3% 0.1% 5.0% Harpacticoida 7.1% 50.0% 14.3% 14.3% 14.3% 10.0% Fontigens 4.0% 0.7% 0.9% 0.9% 0.0% 1.8% 48.9% 31.5% 11.0% 0.3% 0.3% 15.0% Sphaeriidae 0.8% 0.3% 1.6% 1.4% 2.9% 4.1% 10.9% 16.7% 35.4% 9.1% 7.4% 3.3% 2.6% 3.2% 2.8% 20.0% Nematoda 12.0% 62.0% 10.0% 2.0% 8.0% 2.0% 4.0% 25.0% Platyhelminthes 12.4% 15.1% 1.2% 4.5% 2.9% 6.2% 1.4% 0.2% 1.2% 6.8% 12.9% 8.6% 19.9% 11.2% 0.7% 1.2% 1.4% 30.0% Lumbriculidae 3.4% 14.9% 7.9% 1.9% 13.6% 4.1% 16.8% 17.0% 8.4% 0.3% 0.5% 0.8% 0.5% 1.6% 0.3% 2.6% 4.1% 4.4% 35.0% Naididae 1.0% 1.0% 1.0% 1.0% 10.5% 13.3% 27.6% 22.9% 14.3% 1.9% 5.7% 40.0% Lepidostomatidae 1.9% 2.6% 3.2% 64.7% 7.7% 2.6% 2.6% 12.2% 2.6% 45.0% Nemouridae 29.4% 45.1% 11.8% 9.8% 1.9% 3.9% 50.0% Perlidae 75.5% 9.7% 13.4% 1.0% 0.3% 55.0% Dytiscidae 44.4% 5.6% 33.3% 5.6% 5.3% 11.1% 60.0% Dixidae 37.5% 12.5% 12.5% 25.0% 12.5% 65.0% Tanypodinae 10.6% 2.3% 3.4% 1.0% 1.5% 12.9% 9.3% 7.7% 2.1% 1.8% 1.8% 40.2% 0.5% 0.3% 2.8% 0.3% 70.0% Diamesinae 3.0% 4.1% 2.2% 1.9% 8.1% 31.4% 34.7% 4.7% 2.7% 0.7% 4.8% 1.5% 75.0% Ceratopogoninae 11.1% 19.4% 27.8% 13.9% 11.1% 8.3% 5.6% 18.2% 80.0% Hexatoma 13.5% 5.4% 16.2% 21.6% 2.7% 10.8% 2.7% 2.7% 2.7% 2.7% 5.1% 16.2% 2.7% 85.0% Ormosia 22.2% 11.1% 33.3% 22.2% 11.1% 90.0% Pseudolimnophila 4.3% 8.7% 8.7% 26.1% 8.7% 4.3% 20.7% 4.3% 34.8% 95.0% Tipula 27.3% 9.1% 27.3% 9.1% 27.3% 15.4% 100.0% A heat map was generated showing where the common taxa are found across all samples and seasons. 27 taxa are omitted from this heat map as they were found in less than 5 of the 63 samples. The values shown are the relative abundance of taxa i in one sample based on the total count of taxa i in all samples. A scale bar is shown on the right side. Green indicates a low abundance while red indicates a high abundance.

Table 5: Unique Taxa for Each Study Site.

North Seep Upper Seep Lower Seep Pimmit Run Seep C Valley Trail Spring Valley Trail Stream Lumbriculidae 279 Caecidotea 826 Sphaeriidae 547 Caecidotea 1288 Fontigens 3280 Gammarus 147 Perilidae 247 Diamesinae 302 Lumbriculidae 452 Platyhelminthes 121 Gammarus 899 Lumbriculidae 120 Platyhelminthes 120 Lumbriculidae 209 Caecidotea 289 Stygobromus 56 Sphaeriidae 172 Sphaeriidae 75 Diamesinae 68 Fontigens 202 Diamesinae 287 Lumbriculidae 17 Tanypodinae 158 Diamesinae 46 Caecidotea 64 Crangonyx 151 Fontigens 98 Tanypodinae 14 Platyhelminthes 133 Fontigens 23 Tanypodinae 63 Lepidostomatidae 117 Tanypodinae 74 Gyro 8 Lumbriculidae 25 Dicranota 16 Nemouridae 44 Sphaeriidae 73 Naididae 68 Tipula 4 Diamesinae 25 Pseudolimnophila 15 Nematoda 42 Tanypodinae 60 Perilidae 42 Veliidae 3 Lepidostomatidae 23 Tanypodinae 13 Sphaeriidae 24 Platyhelminthes 57 Ostracod 40 Harpacticoida 2 Ostracod 12 Hydropsychidae 11 Leptophlebiidae 14 Naididae 26 Ceratopogoninae 19 Ancylidae 2 Cordulegastridae 5 Platyhelminthes 11 Lepidostomatidae 12 Hexatoma 14 Stygobromus 17 Perilidae 1 Ceratopogoninae 5 Caecidotea 10 Crangonyx 9 Dytiscidae 8 Platyhelminthes 12 Dytiscidae 1 Caecidotea 4 Hexatoma 9 Corydalidae 8 Pseudolimnophila 8 Harpacticoida 9 Hexatoma 1 Cyclopoida 3 Ceratopogoninae 8 Odontoceridae 7 Ceratopogoninae 7 Dytiscidae 7 Nemouridae 1 Harpacticoida 2 Naididae 6 Hexatoma 7 Ormosia 6 Hexatoma 6 Nematoda 2 Brachycentridae 6 Sminthuridae 7 Leuctridae 4 Nematoda 5 Naididae 2 Limnephilidae 5 Hydropsychidae 4 Dasyhelea 4 Nemouridae 5 Ptychopteridae 2 Ormosia 3 Dixidae 4 Tipula 4 Sminthuridae 5 Hexatoma 2 Dytiscidae 3 Ceratopogoninae 4 Perlodidae 2 Lepidostomatidae 4 Limnephilidae 1 Nemouridae 2 Naididae 3 Ephydridae 2 Crangonyx 3 Libellulidae 1 Molanna 2 Limnephilidae 3 Harpacticoida 1 Molanna 2 Pseudolimnophila 1 Tipula 2 Molanna 3 Nematoda 1 Dixidae 2 Sciomyzidae 1 Leptophlebiidae 1 Pseudolimnophila 3 Dixidae 1 Pseudolimnophila 2 Libellulidae 1 Tipula 3 Tabanidae 1 Sciomyzidae 1 Dixidae 1 Tabanidae 2 Culicidae 1 Sciomyzidae 1 Dicranota 2 Syrphidae 1 Cordulegastridae 1 Brachycentridae 1 Stratiomyidae 1 Dasyhelea 1 Veliidae 1 Total 1049 Total 2089 Total 1996 Total 1519 Total 4758 Total 538 Unique taxa and individual counts for each study site are shown above including the total number of individuals counted at that site.

Rank Abundances of All Sites, All Seasons

1 North Seep Upper Seep Lower Seep 0.1 Pimmit Run Seep C Valley Trail Spring Valley Trail Stream

0.01 Relative Relative Abundance 0.001

0.0001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Abundance Rank

Figure 6: Rank-Abundance Curves for All Study Sites and All Seasons. Organism counts were totaled across all seasons and given a rank based on their relative abundance. Relative abundances are plotted on a log base 10 scale.

A Simpson's Index of Diversity

10.00

8.00 Winter Spring 6.00 Summer

D, All D, 4.00

2.00

0.00

B 10.00

8.00

6.00

4.00 D, Insects D, 2.00

0.00

C 10.00 8.00

6.00

Insets - 4.00

D, Non D, 2.00 0.00 North Seep Upper Seep Lower Seep Pimmit Run Valley Trail Valley Trail Seep C Spring Stream

Figure 7: Bar Charts of Simpson’s Diversity Coefficients from the Jackknifed Samples. Standard error bars are included. Winter samples are shown as a squiggle pattern. Spring samples are shown as a plaid pattern. Summer samples are shown as a diagonal line. Coefficients for all organisms, insects only, and non-insects only are shown as A, B, and C.

A Shannon's Index of Diversity

4.00 Winter 3.00 Spring Summer

2.00 H', H', All

1.00

0.00

B 4.00

3.00

2.00

1.00 H', H', Insects

0.00

C 4.00

3.00

2.00

Insects - 1.00

H', H', Non 0.00 North Seep Upper Seep Lower Seep Pimmit Run Valley Trail Valley Trail Seep C Spring Stream

Figure 8: Bar Charts of Shannon’s Diversity Coefficients from the Jackknifed Samples. Standard error bars are included. Winter samples are shown as a squiggle pattern. Spring samples are shown as a plaid pattern. Summer samples are shown as a diagonal line. Coefficients for all organisms, insects only, and non-insects only are shown as A, B, and C.

Community Comparisons

To establish similarity between community composition of the study sites, these similarity indices were performed: Jaccard’s coefficient of similarity (Js), Sorenson’s coefficient of similarity (Ss), Bray-Curtis measure of similarity (BCs), and percent similarity (Ps). Intra-site similarities in the same season were not performed. To identify if samples were similar, an arbitrary threshold value of similarity was created for each index used. This was done by taking the average of the similarity coefficients of the same site for each season comparison (seen in

Table 6), then averaging this across all sites. This was performed for each index and the results are listed in Table 7. Sites were deemed similar or slightly similar if they were within one or two standard deviations of the average, where they would be either green or yellow, respectively.

When examining the intra-site similarities, based on Jaccard’s and Sorenson’s index, the winter to spring comparisons were often less similar than the winter to summer comparisons, and the spring to summer comparisons were often less similar than either the winter to spring or winter to summer comparisons. Conversely, based on the Bray-Curtis measure and percent similarity, the winter-spring samples were often more similar that the winter to summer samples were (Table 6).

Since the indices were largely similar for the season to season comparisons

(Supplemental Table 2-4), only the Jaccard’s Index and Bray-Curtis Index were shown. From the

Jaccard’s coefficient, it is seen that the North Seep, Upper Seep, and Lower Seep sites were found to be similar to each other based on presence absence data and each of these seepage spring sites were slightly similar to the Valley Trail Spring and Valley Trail Stream, which were similar to each other (Table 8). Based on organism abundance and presence, the Bray-Curtis coefficient shows that Upper Seep is slightly similar to both Lower Seep and Pimmit Run Seep C

(Table 8), likely due to the high number of C. kenki found in each study site (Table 5). 30

Table 6: Similarity Coefficients Between Seasons.

Winter- Winter- Spring- Standard Average spring summer summer deviation 퐉퐬 North Seep 0.54 0.65 0.52 0.57 0.07 퐒퐬 North Seep 0.70 0.79 0.68 0.72 0.06 퐁퐂퐬 North Seep 0.39 0.48 0.44 0.44 0.05 퐏퐬 North Seep 0.42 0.49 0.60 0.50 0.09 퐉퐬 Upper Seep 0.50 0.64 0.48 0.54 0.09 퐒퐬 Upper Seep 0.67 0.78 0.65 0.70 0.07 퐁퐂퐬 Upper Seep 0.57 0.48 0.65 0.57 0.09 퐏퐬 Upper Seep 0.63 0.49 0.64 0.59 0.08 퐉퐬 Lower Seep 0.70 0.45 0.58 0.58 0.12 퐒퐬 Lower Seep 0.82 0.63 0.74 0.73 0.10 퐁퐂퐬 Lower Seep 0.59 0.54 0.46 0.53 0.07 퐏퐬 Lower Seep 0.59 0.54 0.49 0.54 0.05 퐉퐬 Pimmit Run Seep C 0.33 0.33 0.67 0.44 0.19 퐒퐬 Pimmit Run Seep C 0.50 0.50 0.80 0.60 0.17 퐁퐂퐬 Pimmit Run Seep C 0.81 0.74 0.62 0.72 0.10 퐏퐬 Pimmit Run Seep C 0.96 0.89 0.92 0.92 0.04 퐉퐬 Valley Trail Spring 0.50 0.35 0.44 0.43 0.08 퐒퐬 Valley Trail Spring 0.67 0.52 0.61 0.60 0.07 퐁퐂퐬 Valley Trail Spring 0.78 0.34 0.48 0.53 0.22 퐏퐬 Valley Trail Spring 0.91 0.89 0.91 0.90 0.01 퐉퐬 Valley Trail Stream 0.38 0.60 0.43 0.47 0.12 퐒퐬 Valley Trail Stream 0.55 0.75 0.61 0.63 0.11 퐁퐂퐬 Valley Trail Stream 0.61 0.62 0.52 0.58 0.05 퐏퐬 Valley Trail Stream 0.67 0.60 0.53 0.60 0.07 For each study site the following is listed: Jaccard’s coefficient of similarity (J푠), Sorenson’s coefficient of similarity (Ss), Bray-Curtis measure of similarity (BCs), and percent similarity (Ps). Intra-site similarity coefficients are shown comparing the seasons. Winter to spring, winter to summer, and spring to summer comparisons are performed.

Table 7: Threshold Similarity Values.

Standard Index Threshold Deviation 퐉퐬 0.51 0.11 퐒퐬 0.66 0.10 퐁퐂퐬 0.56 0.09 퐏퐬 0.68 0.06 Threshold values established from averages similarities between seasons across all study sites used for identifying similarity.

Table 8: Inter-Site Similarity Coefficients.

North Upper Lower Pimmit Valley Valley Seep Seep Seep Run Seep Trail Trail J_s BC_s C Spring Main Stream North Seep 0.28 ± 0.36 ± 0.15 ± 0.08 ± 0.32 ± 0.08 0.01 0.01 0.01 0.13 Upper Seep 0.38 ± 0.39 ± 0.51 ± 0.11 ± 0.22 ± 0.06 0.18 0.12 0.02 0.09 Lower Seep 0.38 ± 0.48 ± 0.19 ± 0.13 ± 0.25 ± 0.12 0.09 0.06 0.05 0.02 Pimmit Run Seep C 0.21 ± 0.21 ± 0.26 ± 0.03 ± 0.05 ± 0.02 0.05 0.04 0.02 0.01 Valley Trail Spring 0.29 ± 0.37 ± 0.42 ± 0.18 ± 0.14 ± 0.08 0.07 0.12 0.03 0.05 Valley Trail Main 0.36 ± 0.32 ± 0.37 ± 0.19 ± 0.43 ± Stream 0.12 0.10 0.05 0.04 0.07 The average similarity coefficients for Jaccard’s Index and Bray-Curtis Index are shown across the three seasons ± the standard deviation. Jaccard’s similarity coefficient is shown on the left side of the matrix while the Bray-Curtis similarity coefficient is seen on the right side of the matrix. Sorenson’s and Percent similarity are provided in appendix A.

Trophic Analysis

Trophic analysis using dual abundance δ13C and δ15N of the two study sites, Pimmit Run

Seep C and Lower Seep, yielded similar results, with the major difference being the leaf and soil signatures. Pimmit Run Seep C is dominated by maple leaves whereas Lower Seep 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 δ13C and δ15N signatures of the maple leaf types analyzed at Pimmit Run Seep C are consistent with documented findings of C3 leaves. However, our samples do not follow the seasonal variation of δ13C ranging from -22‰ to

-28‰ in early spring and late fall in fresh leaf samples (Lowdon & Dyck, 1973), as our samples were leaves from the fall season remaining in the site. Seasonal variation of δ15N is observed, as the maple leaf samples become more enriched in nitrogen-15, leading to a higher δ15N ratio, from winter, through spring, to summer, ranging from 1‰ to 3‰ for Pimmit Run Seep C. The isotopic signatures of the beech leaves from Lower Seep are also consistent with documented findings and are similar to documented seasonal variations (Fotelli et al., 2003) with a lower

15 δ N ratio, compared to maple leaf, as a result of nitrogen fixation (Mariotti, 1983; Unkovich,

2013).

The leaf-litter-soil continuum is well represented in these models and follows the increased enrichment patterns related to increased depth (Balesdent et al., 1993). Some of our data shows the dry leaf having δ13C and δ15N than wet leaf which contradicts the idea that as the leaves decay, they will become more enriched in carbon-13 and nitrogen-15, increasing the δ13C and δ15N ratio. 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 having a

lower δ13C ratio than the fall leaves (Balesdent et al., 1993). The suspended particulate organic matter (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 of our models containing data for them. The glass fiber filters were more enriched in nitrogen-15 than the leaf litter and soil, with samples ranging from 1-5‰ δ15N, but had roughly the same δ13C signature.

The data collected showed similar isotope signatures for the organisms over the three seasons, winter, spring, and summer, so the seasons were averaged and only the average values across all seasons are shown with standard deviations as error bars. The models produced from the data show that the Tipula larva are within the both the δ13C and δ15N fractionation ranges (1-

2‰ and 3-4‰, respectively) of the soil and leaf litter in all of the models with Tipula sampled, except for Lower Seep in the winter season where it is not within the δ13C fractionation range

(Supplemental Figure 4). The aquatic Lumbricidae worms, Lumbriculidae, are within the δ15N fractionation range and just outside the δ13C fractionation range of the soil and leaf samples. The isopod, C. kenki, is within both δ13C and δ15N fractionation ranges of Lumbriculidae. For the amphipod, S. tenuis, the data provides a mixed message. In Pimmit Run Seep C, S. tenuis have a higher δ15N ratio than any other samples but have a slightly lower δ13C ratio compared to C. kenki whereas in Lower Seep they have higher ratios of both δ13C and δ15N than any other organism. In Pimmit Run Seep C, S. tenuis is within the fractionation ranges of the glass fiber filters, but not in Lower Seep (Figures 9 & 10). In Lower Seep in the summer S. tenuis was not found but another amphipod, C. shoemakeri was found in its place. It had a similar isotopic profile as S. tenuis in the Lower Seep sites, but with a slightly lower ratio of both δ13C and δ15N

(Figure 10). When sampled, the Pseudolimnophila and Platyhelminthes had similar δ15N signatures as C. kenki with a lower δ13C raio by about 1-2‰.

These fractionation ranges show that the leaf and soil samples make up 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 which are a potential source of carbon and nitrogen for C. shoemakeri. It is unclear what could be the major source of carbon and nitrogen for S. tenuis, Pseudolimnophila, and the Platyhelminthes. Trophic positions are relatively clear for all organisms sampled.

Pimmit Run Seep C

10 Terrestrial Earthworm Stygobromus (1) tenuis (32) 8 Flatworm (1) Pseudolimnophila (1) Caecidotea kenki (37) 6 GFF (10)

SPOM (8) 4 Lumbriculidae (13)

Wet Maple Leaves (15) 15N δ 2 Dry Maple Leaves (15) Tipula (3) 0

Soil (15) DOC (8) -2

-4 -35 -33 -31 -29 -27 -25 -23 δ13C Figure 9: Dual Abundance Isotope Signatures in Pimmit Run Seep C. Data points are averaged across all seasons. Each data point is labeled with the sample number in parentheses and with error bars showing standard deviation. Some error bars are omitted as there is only one sample. Individual seasons are shown in appendix A. GFF = Glass Fiber Filters. SPOM = Suspended Particulate Organic Matter. DOC = Dissolved Organic Carbon.

Lower Seep 10 Stygobromus tenuis (6) 8 Pseudolimnophila (1) Crangonyx shoemakeri (2) 6 GFF (5) Caecidotea 4 Soil (15) kenki (15)

15N SPOM (11)

δ Lumbriculidae (8) 2 Wet Beech leaves (15) Tipula (2) 0

DOC (6) -2 Dry Beech leaves (15)

-4 -35 -33 -31 -29 -27 -25 -23 δ13C

Figure 10: Dual Abundance Isotope Signatures in Lower Seep. Data points are averaged across all seasons. Each data point is labeled with the sample number in parentheses and with error bars showing standard deviation. Some error bars are omitted as there is only one sample. Individual seasons are shown in appendix A. GFF = Glass Fiber Filters. SPOM = Suspended Particulate Organic Matter. DOC = Dissolved Organic Carbon.

Discussion

The study sites that were observed showed a large presence of insect taxa at the seepage springs but they did not make up the majority of the abundance of organisms found. Crustaceans and mollusks made up the majority. Each of the study sites had a very similar community composition between the winter, spring, and summer seasons. The Jaccard’s and Bray-Curtis indices had average similarity values of 0.51 and 0.56, respectively, between the seasons. Among all seasons it was found that each of the study sites in Great Falls, MD were similar to each other, from Jaccard’s coefficient, with high levels of diversity, suggesting that the area supports a healthy watershed environment. The study sites in Pimmit Run had a lower diversity and species

richness but was still found to be similar to the seepage springs in Great Falls, MD based on the

Bray-Curtis coefficient, likely due to the abundance of C. kenki. This further shows that C. kenki flourishes in the unique seepage spring environments. Unfortunately, the seepage springs did not undergo any dry periods, where they ceased to flow, during our time frame, so we could not get an idea of how the community composition responds to such an event. Understanding this community response would be beneficial to understanding the basic biology of these seepage springs, and how we should react to drying conditions, especially when these environments house the only endangered species in Washington, D.C..

It is virtually impossible to identify if the species are groundwater species or surface species because we cannot visualize them entering and leaving the hypotelminorheic habitats, leaving us forced to make assumptions based on morphology alone. Stygobromus, some

Caecidotea, and some Crangonyx species are known to occupy the underground hypotelminorheic habitat (Culver et al., 2006; Keany et al., 2018). Caecidotea kenki was found in every seepage spring normally in a large presence except for the Valley Trail Spring and

Valley Trail Stream sites. When found, they would be in very few numbers and only ever as a juvenile specimen, never an adult. This indicates that C. kenki could be using the hypotelminorheic habitat or that the juvenile organisms are traversing through the underground and, by chance alone, end up at the Valley Trail Spring site. The small presence in the Valley

Trail Stream site is likely due to upstream discharge from Upper Seep. Another possibility is that the C. kenki could simply be eaten by the G. minus as they grow into an adult and that is why they were never observed. Only S. tenuis were collected at Lower Seep while during later sampling events at the same site we collected adult specimens of C. shoemakeri and G. minus.

The amphipods cannot grow to adult in this amount of time, so it is assumed they traversed through the hypotelminorheic habitat that is potentially connecting the seepage springs.

Whether organisms are using the hypotelminorheic habitat or not, they can still be classified as seep specialists, in the sense that they rely on the unique water chemistry and subterranean environment that this habitat has to offer. We attempted to identify potential specialist species by examining if they had a persistent presence equal to the that of the stygobiont S. tenuis. We identified 7 non-insect taxa that could be deemed specialists:

Harpacticoida, F. bottimeri, Sphaeriidae, Nematoda, Platyhelminthes, Lumbriculidae, Naididae, and 9 insect taxa: Lepidostomatidae, Tanypodinae, Diamesinae, Hexatoma, Ormosia,

Pseudolimnophila, Tipula, Perlidae and Nemouridae. All other taxa found were in small numbers and inconsistently in the study sites and are assumed to be generalists, or occupying the site based on chance alone. The stone flies, Perlidae and Nemouridae, were found in juvenile stages and were found heavily in the winter and spring seasons, suggesting they were there as a result of breeding events. Stanford & Gauffin (1974) show that two stonefly communities of the family

Chloroperlidae spend their entire nymphal cycles in the subterranean habitats of two rivers, supporting the idea that the stoneflies discovered in the seepage springs could be specialists to the hypotelminorheic habitat. Although the other taxa that were identified as potential specialists were found at a similar rate as the stygobiont, and at the seepage window, they are still found in the seepage spring run and in either the spring or stream habitats. Whereas true seep specialists that utilize the hypotelminorheic, like S. tenuis are found only near the seepage window and in seepage springs only.

We performed dual abundance δ13C and δ15N isotope analysis for S. tenuis, C. shoemakeri, C. kenki, Platyhelminthes, Lumbriculidae, Pseudolimnophila, and Tipula. Others

were not analyzed either for them not having enough mass for computation or being found at a high rate after the initial sampling events for trophic analysis. We discovered from the models created from our data that the Lumbriculidae worms and Tipula larvae likely occupy the same

FFG, are obtaining their energy from soil and leaf litter, and are one trophic position higher than the baseline. 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 not over time. The isopods, C. kenki, are likely obtaining their energy from some combination of the

Lumbriculidae worms and another organism that was not sampled and are assumed to be two trophic positions higher than the base. Stygobromus tenuis and C. shoemakeri seem to be at different trophic positions, with S. tenuis being higher than C. shoemakeri. Crangonyx 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 believed that they could be eating the copepods, but an extensive search for copepods 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 functional feeding group and trophic position at C. kenki, but our data does not distinguish where they are getting their energy from. Sampling of more insect taxa could provide this information.

Our efforts to identify the community composition and food web dynamics of these unique seepage spring habitats was informative, but more research needs to be done before being conclusive. There was a clear flow of energy but it is still unclear what S. tenuis is obtaining its energy from. It would be useful to analyze F. bottimeri and Sphaeriidae for trophic analysis, as mollusks are known to create a baseline over a temporal plane, indicating the base over seasons with less variation, opposed to leaf litter which shows a baseline at an instant in time, which can

vary among seasons (Post, 2002). Identifying a way to remove the shell without destroying the organism is needed for this to work. Additionally, increasing sample size to 6 samples per season instead of 3 would be beneficial in producing more quantitative samples and subsequent identification of seep specialists vs. generalists.

APPENDIX A

Table 9: Biodiversity Estimates.

Valley Pimmit North Upper Lower Pimmit Valley Trail Trail Run Seep Seep Seep Run Seep C Spring Stream Stream D, all Winter 8.9 ± 1.10 3.6 ± 0.84 5.5 ± 0.97 1.3 ± 0.04 2.0 ± 0.49 5.1 ± 3.41 1.7 ± 1.15 Spring 4.4 ± 0.20 4.2 ± 0.51 6.0 ± 0.65 1.3 ± 0.08 1.8 ± 0.62 8.5 ± 1.70 1.9 ± 0.19 Summer 4.5 ± 3.47 5.5 ± 1.07 3.3 ± 0.69 1.6 ± 0.28 1.7 ± 0.36 5.6 ± 4.32 3.6 ± 1.27 D,

insects Winter 5.1 ± 1.22 1.5 ± 1.12 1.8 ± 1.03 1.7 ± 0.92 1.4 ± 0.37 6.5 ± 0.43 1.7 ± 1.15 Spring 1.8 ± 0.71 2.3 ± 0.50 1.3 ± 1.58 1.7 ± 0.25 1.8 ± 1.37 4.8 ± 3.52 1.9 ± 0.19 Summer 5.7 ± 0.39 2.0 ± 0.60 1.6 ± 4.73 2.9 ± 0.89 3.1 ± 0.21 6.8 ± 0.64 3.6 ± 1.27 D, non-

insects Winter 4.3 ± 0.58 2.7 ± 0.91 4.6 ± 0.67 1.3 ± 0.04 1.7 ± 0.34 3.7 ± 1.88 0.0 ± 0.00 Spring 2.7 ± 0.89 3.2 ± 0.43 3.9 ± 0.35 1.3 ± 0.08 1.8 ± 0.48 4.4 ± 0.60 0.0 ± 0.00 Summer 1.7 ± 1.21 3.1 ± 2.12 2.7 ± 0.50 1.5 ± 0.31 1.7 ± 0.39 3.0 ± 1.95 0.0 ± 0.00 H', all Winter 2.4 ± 0.12 1.7 ± 0.14 2.1 ± 0.14 0.5 ± 0.04 1.1 ± 0.27 2.2 ± 0.75 0.0 ± 0.00 Spring 2.0 ± 0.13 1.9 ± 0.09 2.0 ± 0.24 0.6 ± 0.06 1.0 ± 0.33 2.5 ± 0.15 1.0 ± 0.13 Summer 2.2 ± 0.5 2.2 ± 0.14 1.8 ± 0.15 0.9 ± 0.18 0.9 ± 0.18 2.3 ± 0.47 1.8 ± 0.96 H',

insects Winter 2.0 ± 0.21 0.9 ± 0.63 1.0 ± 0.39 0.8 ± 0.73 0.7 ± 0.28 2.4 ± 0.25 0.0 ± 0.00 Spring 1.3 ± 0.42 1.4 ± 0.21 0.9 ± 0.78 0.9 ± 0.27 1.3 ± 0.15 2.2 ± 0.58 1.0 ± 0.13 Summer 2.1 ± 0.11 1.2 ± 0.51 1.7 ± 0.75 1.5 ± 0.28 1.7 ± 0.12 2.2 ± 0.20 1.8 ± 0.96 H', non-

insects Winter 1.6 ± 0.14 1.3 ± 0.31 1.8 ± 0.07 0.5 ± 0.04 0.9 ± 0.26 1.6 ± 0.49 0.0 ± 0.00 Spring 1.3 ± 0.28 1.4 ± 0.14 1.5 ± 0.02 0.5 ± 0.08 0.9 ± 0.21 1.6 ± 0.16 0.0 ± 0.00 Summer 1.1 ± 0.59 1.7 ± 0.47 1.4 ± 0.15 0.7 ± 0.26 0.8 ± 0.22 1.4 ± 0.42 0.0 ± 0.00 Biodiversity estimates, as a result of jackknifing, are listed for each study site and each season. Estimates are separated into three categories: all organisms, insects, and non-insects. For each, Simpson’s index of diversity (D) and Shannon’s index of diversity (H’) is listed with ± of the standard deviation. Standard error can be calculated as the standard deviation divided by √3 .

Table 10: Similarity Coefficients in Winter.

Pimmit Valley Valley North Upper Lower Coefficient in Winter Run Trail Trail Seep Seep Seep Seep C Spring Stream

퐉퐬 Upper Seep 0.44 - - - - -

퐒퐬 Upper Seep 0.61 - - - - - 퐁퐂퐬 Upper Seep 0.23 - - - - - 퐏퐬 Upper Seep 0.29 - - - - - 퐉퐬 Lower Seep 0.26 0.43 - - - -

퐒퐬 Lower Seep 0.41 0.60 - - - - 퐁퐂퐬 Lower Seep 0.35 0.23 - - - - 퐏퐬 Lower Seep 0.39 0.26 - - - - 퐉퐬 Pimmit Run Seep C 0.18 0.16 0.25 - - -

퐒퐬 Pimmit Run Seep C 0.31 0.27 0.40 - - - 퐁퐂퐬 Pimmit Run Seep C 0.22 0.64 0.19 - - - 퐏퐬 Pimmit Run Seep C 0.25 0.52 0.18 - - - 퐉퐬 Valley Trail Spring 0.28 0.43 0.55 0.15 - -

퐒퐬 Valley Trail Spring 0.43 0.61 0.71 0.26 - - 퐁퐂퐬 Valley Trail Spring 0.08 0.12 0.12 0.02 - - 퐏퐬 Valley Trail Spring 0.13 0.24 0.18 0.04 - - 퐉퐬 Valley Trail Stream 0.24 0.33 0.43 0.16 0.43 -

퐒퐬 Valley Trail Stream 0.39 0.50 0.60 0.27 0.61 - 퐁퐂퐬 Valley Trail Stream 0.22 0.12 0.25 0.03 0.08 - 퐏퐬 Valley Trail Stream 0.23 0.17 0.46 0.05 0.31 - 퐉퐬 Pimmit Run Stream 0.05 0.06 0.07 0.00 0.06 0.06 퐒퐬 Pimmit Run Stream 0.10 0.12 0.13 0.00 0.11 0.12 퐁퐂퐬 Pimmit Run Stream 0.01 0.00 0.00 0.00 0.00 0.01 퐏퐬 Pimmit Run Stream 0.16 0.01 0.07 0.00 0.06 0.01 Similarity coefficients for all study sites in the winter seasons are shown above. Js, Ss, BCs, and Ps refer to Jaccard’s Index, Sorenson’s Index, Bray-Curtis Index, and percent similarity, respectively. The cells shaded green were within one standard deviation of the threshold value and samples in yellow were within two standard deviations of the threshold values outlined in Table 4.

Table 11: Similarity Coefficients in Spring.

Pimmit Valley Valley North Upper Lower Coefficient in Spring Run Trail Trail Seep Seep Seep Seep C Spring Stream

퐉퐬 Upper Seep 0.33 - - - - - 퐒퐬 Upper Seep 0.50 - - - - - 퐁퐂퐬 Upper Seep 0.37 - - - - - 퐏퐬 Upper Seep 0.37 - - - - - 퐉퐬 Lower Seep 0.39 0.42 - - - - 퐒퐬 Lower Seep 0.56 0.59 - - - - 퐁퐂퐬 Lower Seep 0.36 0.59 - - - - 퐏퐬 Lower Seep 0.35 0.59 - - - - 퐉퐬 Pimmit Run Seep C 0.22 0.23 0.30 - - -

퐒퐬 Pimmit Run Seep C 0.36 0.37 0.47 - - - 퐁퐂퐬 Pimmit Run Seep C 0.12 0.41 0.25 - - - 퐏퐬 Pimmit Run Seep C 0.11 0.44 0.23 - - - 퐉퐬 Valley Trail Spring 0.38 0.36 0.38 0.21 - -

퐒퐬 Valley Trail Spring 0.56 0.53 0.55 0.35 - - 퐁퐂퐬 Valley Trail Spring 0.09 0.09 0.09 0.05 - - 퐏퐬 Valley Trail Spring 0.06 0.13 0.07 0.04 - - 퐉퐬 Valley Trail Stream 0.48 0.31 0.32 0.17 0.50 -

퐒퐬 Valley Trail Stream 0.65 0.47 0.49 0.30 0.67 - 퐁퐂퐬 Valley Trail Stream 0.27 0.30 0.24 0.05 0.16 - 퐏퐬 Valley Trail Stream 0.32 0.39 0.52 0.07 0.32 - 퐉퐬 Pimmit Run Stream 0.08 0.11 0.09 0.08 0.13 0.11

퐒퐬 Pimmit Run Stream 0.15 0.19 0.17 0.14 0.24 0.19 퐁퐂퐬 Pimmit Run Stream 0.02 0.02 0.01 0.01 0.00 0.04 퐏퐬 Pimmit Run Stream 0.07 0.06 0.08 0.01 0.01 0.09 Similarity coefficients for all study sites in the spring seasons are shown above. Js, Ss, BCs, and Ps refer to Jaccard’s Index, Sorenson’s Index, Bray-Curtis Index, and percent similarity, respectively. The cells shaded green were within one standard deviation of the threshold value and samples in yellow were within two standard deviations of the threshold values outlined in Table 4.

Table 12: Similarity Coefficients in Summer.

Pimmit Valley Valley North Upper Lower Coefficient in Summer Run Trail Trail Seep Seep Seep Seep C Spring Stream

퐉퐬 Upper Seep 0.36 - - - - -

퐒퐬 Upper Seep 0.53 - - - - - 퐁퐂퐬 Upper Seep 0.24 - - - - - 퐏퐬 Upper Seep 0.34 - - - - - 퐉퐬 Lower Seep 0.50 0.58 - - - -

퐒퐬 Lower Seep 0.67 0.74 - - - - 퐁퐂퐬 Lower Seep 0.37 0.35 - - - - 퐏퐬 Lower Seep 0.36 0.35 - - - - 퐉퐬 Pimmit Run Seep C 0.22 0.25 0.22 - - -

퐒퐬 Pimmit Run Seep C 0.36 0.40 0.36 - - - 퐁퐂퐬 Pimmit Run Seep C 0.11 0.48 0.13 - - - 퐏퐬 Pimmit Run Seep C 0.12 0.38 0.12 - - - 퐉퐬 Valley Trail Spring 0.22 0.30 0.33 0.18 - -

퐒퐬 Valley Trail Spring 0.36 0.47 0.50 0.30 - - 퐁퐂퐬 Valley Trail Spring 0.07 0.12 0.19 0.02 - - 퐏퐬 Valley Trail Spring 0.08 0.12 0.19 0.01 - - 퐉퐬 Valley Trail Stream 0.36 0.33 0.36 0.24 0.37 - 퐒퐬 Valley Trail Stream 0.53 0.50 0.53 0.38 0.54 - 퐁퐂퐬 Valley Trail Stream 0.48 0.24 0.27 0.06 0.17 - 퐏퐬 Valley Trail Stream 0.55 0.28 0.50 0.06 0.25 - 퐉퐬 Pimmit Run Stream 0.05 0.10 0.11 0.08 0.00 0.06 퐒퐬 Pimmit Run Stream 0.10 0.17 0.19 0.15 0.00 0.11 퐁퐂퐬 Pimmit Run Stream 0.03 0.01 0.01 0.02 0.00 0.01 퐏퐬 Pimmit Run Stream 0.06 0.08 0.02 0.02 0.00 0.01 Similarity coefficients for all study sites in the summer seasons are shown above. Js, Ss, BCs, and Ps refer to the Jaccard’s Index, Sorenson’s Index, Bray-Curtis Index, and percent similarity, respectively. The cells shaded green were within one standard deviation of the threshold value and samples in yellow were within two standard deviations of the threshold values outlined in Table 4.

Pimmit Run Seep C, Winter. 01/16/2017 13 11 Glass Fiber Filters 9 Wet Maple leaves 7 Dry Maple Leaves

5 Soil 15N

δ 3 Caecidotea kenki 1 Stygobromus tenuis -1 Terrestrial Lumbricidae -3 Aquatic Lumbricidae -5 -35 -33 -31 -29 -27 -25 -23 δ13C

Figure 11: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Winter Season in Pimmit Run Seep C. Pimmit Run Seep C, Spring. 05/08/2017 13 11 Glass Fiber Filter 9 Wet Maple leaves Dry Maple leaves 7 Soil 5

Caecidotea kenki 15N

δ 3 Stygobromus tenuis 1 Aquatic Lumbricidae -1 SPOM -3 -5 -35 -33 -31 -29 -27 -25 -23

δ13C Figure 12: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Spring Season in Pimmit Run Seep C.

Pimmit Run Seep C, Summer. 09/07/2018 13 11 Wet Maple leaves Dry Maple leaves 9 Soil 7 Caecidotea kenki 5 Stygobromus tenuis

Aquatic Lumbricidae 15N δ 3 SPOM 1 Pseudolimnophila -1 Flatworm Tipula -3 DOC -5 -35 -33 -31 -29 -27 -25 -23 δ13C

Figure 13: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Summer Season in Pimmit Run Seep C. Glass Fiber Filters are not included as they were washed away Lower Seep, Winter. 03/07/2018

13 Wet Beech Leaves

11 Dry Beech Leaves 9 Soil 7 Caecidotea kenki 5

Stygobromus tenuis 15N

δ 3 Aquatic Lumbricidae 1 SPOM -1 Tipula -3 DOC -5 -35 -33 -31 -29 -27 -25 -23 δ13C

Figure 14: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Winter Season in Lower Seep. Glass fiber filters are not included as they were washed away during heavy rains.

Lower Seep, Spring. 06/07/2018

13 Wet Beech Leaves 11 Dry Beech Leaves 9 Soil 7 SPOM 5

Caecidotea Kenki 15N

δ 3 Stygobromus tenuis

1 Aquatic Lumbricidae

-1 Tipula

-3 DOC -5 -35 -33 -31 -29 -27 -25 -23 δ13C

Figure 15: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Spring Season in Lower Seep. Glass fiber filters are not included as they were washed away in heavy rains. Lower Seep, Summer. 09/07/2018 13 Glass Fiber Filters 11 Wet Beech Leaves 9 Dry Beech Leaves 7 Soil 5 Caecidotea kenki

Crangonyx shoemakeri 15N δ 3 Lumbricilidae 1 SPOM -1 Pseudolimnophila DOC -3 -5 -35 -33 -31 -29 -27 -25 -23 δ13C

Figure 16: The δ13C and δ15N Signatures for Each Item or Organism Analyzed During the Spring Season in Lower Seep. 47

Table 13: Raw Isotope Values

Pimmit Run Seep C Winter Sample ID d13C d15N C Amount (ug) N Amount (ug) C/N Glass Fiber Filters -25.75 6.8 122.12 16.99 7.19 Glass Fiber Filters -28.33 6.22 100.13 11.71 8.55 Glass Fiber Filters -28.01 6.57 152.12 18.55 8.20 Glass Fiber Filters -28.71 8.28 140.41 15.00 9.36 Glass Fiber Filters -28.59 7.13 159.06 16.15 9.85 Wet Maple leaves -29.45 1.32 2516.507 65.67474 38.31773 Wet Maple leaves -29.34 1.44 2296.271 61.48036 37.34968 Wet Maple leaves -29.71 1.52 2683.832 80.36412 33.3959 Wet Maple leaves -29.46 1.67 2588.049 71.96846 35.96087 Wet Maple leaves -29.79 1.56 2897.401 82.46375 35.13545 Dry Maple Leaves -30.62 1.04 2435.145 56.76305 42.90017 Dry Maple Leaves -30.78 1.04 2978.772 69.34576 42.95536 Dry Maple Leaves -30.38 1.19 2324.798 57.28713 40.58151 Dry Maple Leaves -30.67 1.05 3286.97 83.51368 39.35846 Dry Maple Leaves -30.76 1.05 2513.387 63.5774 39.53271 Soil -29.05 -1.5 129.0716 9.826751 13.13472 Soil -28.32 0.14 95.3648 7.841559 12.16146 Soil -28.86 1.28 151.7505 10.71495 14.1625 Soil -28.82 0.67 142.2436 11.39419 12.48387 Soil -29.03 -0.8 94.99807 7.841559 12.11469 Caecidotea kenki -23.88 5.21 58.6532 14.4773 4.051392 Caecidotea kenki -25.23 4.75 75.91722 18.65878 4.068714 Caecidotea kenki -25.44 5.93 241.4432 67.24793 3.590344 Caecidotea kenki -26.23 5.9 299.5305 62.00459 4.830779 Caecidotea kenki -26.84 6.17 606.602 126.096 4.810637 Caecidotea kenki -26.18 7.14 773.0101 162.4627 4.758077 Caecidotea kenki -25.22 5.77 204.3099 44.0336 4.639865 Caecidotea kenki -26.73 5.67 431.3063 81.93882 5.26376 Caecidotea kenki -26.97 5.95 659.8953 121.3588 5.437557 Caecidotea kenki -25.56 6.39 212.3258 50.0565 4.241723 Caecidotea kenki -25.06 6.18 560.2747 117.6753 4.761191 Caecidotea kenki -25.21 5.17 61.96039 13.90243 4.456802 Caecidotea kenki -25.79 6.04 663.442 139.2625 4.763969 Caecidotea kenki -25.56 7.09 340.7978 64.10171 5.316516 Caecidotea kenki -24.9 6.52 370.0586 71.96846 5.141954 Caecidotea kenki -26.06 6.39 603.0429 101.3734 5.948731 Caecidotea kenki -24.43 6.3 291.9178 64.10171 4.553978

Stygobromus tenuis -26.22 8.88 261.4329 58.33533 4.481554 Stygobromus tenuis -26.4 9.18 524.55 118.7276 4.418094 Stygobromus tenuis -26.14 8.52 638.5987 138.7356 4.602992 Stygobromus tenuis -26.5 8.73 592.3612 127.1489 4.6588 Stygobromus tenuis -26.62 10.12 923.7697 183.0557 5.046387 Stygobromus tenuis -25.84 8.66 398.9087 93.49154 4.266789 Stygobromus tenuis -26.39 8.88 528.1259 120.3063 4.389845 Stygobromus tenuis -26.82 10.21 962.108 203.1468 4.736023 Stygobromus tenuis -26.05 9.2 398.9087 95.59297 4.172992 Stygobromus tenuis -26.74 10.05 1183.324 247.651 4.778193 Stygobromus tenuis -26.36 8.61 492.3321 109.7851 4.484506 Stygobromus tenuis -26.09 9.47 329.5858 73.5423 4.481582 Stygobromus tenuis -26.47 8.53 570.9771 126.096 4.528115 Stygobromus tenuis -26.82 9.1 549.5653 118.7276 4.62879 Stygobromus tenuis -25.27 8.57 141.8778 34.29723 4.136714 Terrestrial Lumbricidae -27.72 8.85 Aquatic Lumbricidae -26.46 3.4 Aquatic Lumbricidae -25.92 3.95 Aquatic Lumbricidae -26.82 3.86 Aquatic Lumbricidae -26.8 3.73 DOC1 -25.4603 0 DOC3 -24.2809 0 DOC4 -24.986 0 DOC5 -25.4997 0 Spring Sample ID d13C d15N C Amount (ug) N Amount (ug) C/N Glass Fiber Filter -28.79 3.48 80.63387 8.188943 9.846676 Glass Fiber Filter -28.49 3.87 106.3642 18.83221 5.647993 Glass Fiber Filter -27.53 4.73 150.6112 11.92798 12.62671 Glass Fiber Filter -28.48 4.82 78.77429 8.239789 9.560231 Glass Fiber Filter -28.68 4.4 82.04718 6.939842 11.82263 Wet Maple leaves -28.62 2.26 3371.561 125.3624 26.89452 Wet Maple leaves -28.7 2.25 2862.991 104.1221 27.49649 Wet Maple leaves -28.49 2.17 3789.084 139.2749 27.20579 Wet Maple leaves -28.59 2.39 2682.131 97.42982 27.52885 Wet Maple leaves -28.49 2.25 4062.302 144.7719 28.06002 Dry Maple leaves -29.96 2.01 3149.7 98.3746 32.01742 Dry Maple leaves -29.98 1.98 2710.352 83.6545 32.39936 Dry Maple leaves -29.78 2.11 3694.72 112.142 32.94679 Dry Maple leaves -29.93 2.06 3233.293 101.9218 31.72327 Dry Maple leaves -29.95 2.16 3034.326 91.47417 33.1714 Soil -28.15 -0.19 32.86653 2.664989 12.33271 49

Soil -28.08 3.73 44.21619 3.915902 11.29144 Soil -28.23 2.7 42.01162 3.053783 13.75724 Soil -27.81 2.34 28.02866 2.093894 13.3859 Soil -27.29 3.01 70.63695 3.927146 17.98684 Caecidotea kenki -25.3 6.92 529.7263 123.5886 4.286208 Caecidotea kenki -24.77 5.64 742.8398 132.8094 5.593278 Caecidotea kenki -24.83 5.83 711.646 133.6591 5.324338 Caecidotea kenki -24.78 6.13 577.7686 105.5133 5.475791 Caecidotea kenki -25.17 6.06 1000.26 222.1757 4.502113 Caecidotea kenki -25.18 5.91 907.1904 191.8679 4.728203 Caecidotea kenki -25.17 6.14 876.0506 166.2644 5.269021 Caecidotea kenki -25.59 6.18 1059.424 191.3038 5.537916 Caecidotea kenki -24.8 6.12 983.2906 187.2596 5.250949 Caecidotea kenki -24.76 5.81 786.4456 171.1489 4.595095 Caecidotea kenki -24.79 6.57 574.8317 129.5692 4.436482 Caecidotea kenki -25.81 5.9 573.6986 104.7458 5.477054 Caecidotea kenki -25.27 5.92 1053.162 210.7415 4.997413 Caecidotea kenki -25.32 5.57 706.2823 137.6862 5.129652 Caecidotea kenki -25.1 5.85 526.7613 101.0181 5.214524 Stygobromus tenuis -25.41 9.24 412.3439 102.1617 4.036187 Stygobromus tenuis -25.29 9.57 334.6514 83.91891 3.987795 Stygobromus tenuis -25.96 8.38 830.056 180.8084 4.590804 Stygobromus tenuis -26.01 8.94 583.0335 124.4496 4.684896 Stygobromus tenuis -25.04 11.05 281.9136 69.5745 4.051967 Stygobromus tenuis -25.81 9.88 168.8156 41.65424 4.052784 Stygobromus tenuis -25.67 9.14 185.0587 47.11651 3.927684 Stygobromus tenuis -24.46 9.35 113.9261 29.78627 3.824784 Stygobromus tenuis -25.25 10.62 350.2158 78.56577 4.457613 Stygobromus tenuis -25.52 9.25 236.3037 59.67868 3.9596 Stygobromus tenuis -25.83 8.89 401.2556 98.68481 4.066032 Stygobromus tenuis -25.61 8.55 134.0122 35.7302 3.750671 Stygobromus tenuis -25.16 8.63 275.9732 67.85136 4.067319 Stygobromus tenuis -25.2 9.56 324.5034 80.07903 4.05229 Aquatic Lumbricidae -26.27 2.84 579.5211 147.8753 3.918984 Aquatic Lumbricidae -26.13 3.82 3252.635 816.0671 3.985745 Aquatic Lumbricidae -26.93 n.d. 4038.264 958.418 4.213468 Aquatic Lumbricidae -26.49 5.23 3731.523 862.811 4.324844 Aquatic Lumbricidae -26.85 4.78 3719.418 872.9823 4.260588 Aquatic Lumbricidae -26.39 3.91 2912.649 657.3732 4.430739 Aquatic Lumbricidae -26.32 4.73 3227.451 812.609 3.971715 Aquatic Lumbricidae -26.53 3.89 2865.509 632.8709 4.527793 SPOM -29.03 2.59 285.5674 15.61216 18.29135 50

SPOM -29.23 2.71 311.5241 17.45417 17.84812 SPOM -29.18 2.68 271.7578 15.36902 17.68218 SPOM -29.01 2.64 219.7975 12.12249 18.13138 SPOM -29.14 2.79 313.5492 16.18643 19.37112 DOC -28.0807 0 DOC -28.0633 0 Summer Sample ID d13C d15N C amount (ug) N Amount (ug) C/N Wet Maple leaves -29.0539 3.209925 2774.081 58.85818 47.13162 Wet Maple leaves -28.764 3.17719 2951.562 63.19595 46.70492 Wet Maple leaves -29.1707 3.466791 2557.089 56.64541 45.14204 Wet Maple leaves -29.2099 3.670274 2429.172 65.77876 36.92942 Wet Maple leaves -29.5109 3.685739 2229.111 62.06203 35.91748 Dry Maple leaves -27.9839 2.265848 2758.719 43.07323 64.04718 Dry Maple leaves -27.724 2.244336 3700.372 54.89468 67.40857 Dry Maple leaves -28.1523 2.617003 2982.319 65.51193 45.5233 Dry Maple leaves -27.5256 2.351341 2832.329 52.22605 54.23212 Dry Maple leaves -25.248 2.512406 2802.897 44.64952 62.77553 Soil -27.6441 -14.3773 12.5032 1.863215 6.710553 Soil -27.4281 -1.2913 11.0267 2.135148 5.16437 Soil -29.294 1.380047 39.88898 3.195896 12.48131 Soil -27.3143 -0.56441 9.582808 2.09798 4.567634 Soil -27.4168 -1.34501 5.08097 1.633123 3.111199 Caecidotea kenki -25.3365 4.884561 184.5767 33.81508 5.458413 Caecidotea kenki -25.4413 5.537907 429.3224 83.45514 5.144349 Caecidotea kenki -24.8509 5.003745 213.5445 45.69024 4.673745 Caecidotea kenki -25.3978 4.71097 169.095 31.86292 5.306951 Caecidotea kenki -25.7837 5.140515 220.1676 43.05052 5.114168 Stygobromus tenuis -24.8222 6.778376 25.81509 6.652086 3.880751 Stygobromus tenuis -24.9331 8.142083 326.3908 76.70805 4.254974 Stygobromus tenuis -24.85 7.82964 192.8021 49.0053 3.93431 Aquatic Lumbricidae -26.5468 3.539942 3303.488 806.7823 4.094646 Aquatic Lumbricidae -25.7581 2.961897 90.18282 22.84162 3.94818 Aquatic Lumbricidae -26.5301 3.258869 616.0893 153.5616 4.012002 SPOM -29.0144 3.179122 374.9346 20.68846 18.12289 SPOM -29.0085 3.085112 432.1001 23.44605 18.42955 SPOM -29.1182 3.105427 593.2021 31.38659 18.89986 Pseudolimnophila -27.2487 7.147661 375.5495 69.34342 5.415791 Flatworm -26.7823 7.540157 954.1322 183.5935 5.196982 Tipula -27.3102 2.141806 1091.231 209.0953 5.218822 Tipula -27.5126 2.558353 2072.179 391.3061 5.295545 Tipula -27.0468 2.217242 1109.641 259.536 4.275482 51

DOC -25.8036 0 DOC -25.6229 0 Lower Seep Winter Sample ID d13C d15N C amount (ug) N Amount (ug) C/N SPOM -29.7683 1.002145 764.9594 42.21667 18.11984 SPOM -29.7848 0.977611 1682.933 93.80526 17.94071 SPOM -29.7714 1.061047 941.8902 53.05148 17.75427 SPOM -29.8173 1.02282 1539.06 86.16727 17.8613 SPOM -29.7961 1.019487 1941.48 108.498 17.89416 Wet Beech Leaves -33.4951 -0.94986 1828.319 53.39388 34.24211 Wet Beech Leaves -32.2849 -0.80471 3800.079 116.2027 32.70215 Wet Beech Leaves -32.2357 -1.52621 3247.684 97.43462 33.33193 Wet Beech Leaves -32.0005 -0.43494 3271.076 117.9877 27.72388 Wet Beech Leaves -32.615 -0.80109 1781.018 53.19424 33.48142 Dry Beech Leaves -31.9863 -2.26074 3793 73.28365 51.75779 Dry Beech Leaves -32.4302 -2.5015 2927.022 72.16287 40.56133 Dry Beech Leaves -30.5023 -1.20324 2055.179 50.64033 40.58385 Dry Beech Leaves -32.5945 -2.37419 3816.969 109.6844 34.79957 Dry Beech Leaves -31.1751 -2.37455 3394.867 109.257 31.07231 Stygobromus tenuis -26.0746 8.639106 699.5907 166.6206 4.198704 Stygobromus tenuis -25.873 8.028834 948.2765 219.0392 4.329255 Stygobromus tenuis -25.6666 8.697355 558.1565 135.4711 4.120114 Stygobromus tenuis -25.8678 8.61623 773.9936 172.1246 4.496706 Stygobromus tenuis -25.9479 7.741992 362.8103 87.32167 4.154871 Caecidotea kenki -24.5149 4.552774 890.7401 182.3953 4.88357 Caecidotea kenki -27.987 6.119236 1593.383 266.2436 5.984682 Caecidotea kenki -26.6062 4.807548 1151.245 218.371 5.271968 Caecidotea kenki -26.6876 5.346891 786.2831 163.0709 4.821725 Caecidotea kenki -27.6664 5.772156 1053.504 228.0358 4.619904 Tipula -27.1454 4.007019 2237.21 516.2793 4.333333 Aquatic Lumbricidae -26.5513 5.360065 549.3804 137.8436 3.985536 Aquatic Lumbricidae -25.6598 7.307202 256.2544 70.464 3.636672 Soil -29.3638 1.686393 123.2531 8.024684 15.35924 Soil -29.2471 1.37779 91.19934 5.567041 16.38201 Soil -29.0041 1.307738 74.26193 4.846667 15.32227 Soil -29.431 1.421155 146.0977 8.930339 16.3597 Soil -29.1355 1.506187 83.99372 5.442421 15.43315 DOC -29.4504 0 DOC -29.4959 0

Spring Sample ID d13C d15N C amount (ug) N Amount (ug) C/N SPOM -29.5996 1.041324 904.0056 53.37469 25.83606 SPOM -29.6703 0.986555 649.4621 38.7142 30.19646 SPOM -29.4579 1.259287 352.2547 20.94443 63.06952 Wet Beech Leaves -31.0421 -1.25774 2671.243 84.25845 43.23132 Wet Beech Leaves -30.8282 -0.95576 2470.108 83.93531 41.38412 Wet Beech Leaves -31.0077 -0.77325 2278.279 82.31038 35.58744 Wet Beech Leaves -30.9579 -1.11658 2232.738 73.37462 45.64393 Wet Beech Leaves -31.0826 -1.14662 1982.752 63.53229 57.32186 Dry Beech Leaves -30.8974 -3.38975 1989.275 55.69471 73.0585 Dry Beech Leaves -30.7661 -4.01146 2860.932 63.44016 64.42364 Dry Beech Leaves -30.9946 -3.97858 2373.591 60.97641 67.37258 Dry Beech Leaves -30.911 -3.95859 2434.994 61.12497 65.18671 Dry Beech Leaves -31.0067 -3.74719 3303.54 92.64079 42.7916 Caecidotea Kenki -25.979 3.206137 155.8931 30.74696 114.0794 Caecidotea Kenki -25.4906 3.52291 235.4868 50.45987 70.0022 Caecidotea Kenki -25.7408 4.628438 269.2571 50.68985 68.29526 Caecidotea Kenki -26.3362 5.059769 1992.725 368.5683 8.247454 Caecidotea Kenki -26.1119 3.902047 542.8801 93.77855 34.73375 Stygobromus tenuis -25.1422 7.227942 128.1337 30.16841 191.1276 Aquatic Lumbricidae -26.3683 2.754761 1676.697 384.7333 8.71615 Aquatic Lumbricidae -26.7131 2.699529 828.6783 175.3293 17.72404 Aquatic Lumbricidae -26.912 3.122955 549.0963 114.6418 26.94189 Tipula -28.8032 1.054638 369.8719 84.37588 49.31574 Soil -29.3818 1.341732 91.43913 6.08131 17.34933 Soil -29.4064 0.971368 71.44341 4.859422 24.05788 Soil -29.3298 1.210117 94.01027 6.376532 15.07824 Soil -29.335 0.729748 68.99661 4.910592 28.10114 Soil -29.2884 1.300089 95.45671 6.514136 16.4855 DOC -28.5217 0 DOC -28.6735 0 Summer Sample ID d13C d15N C amount (ug) N Amount (ug) C/N Glass Fiber Filters -29.3559 2.412404 621.2923 39.46394 Glass Fiber Filters -29.2577 2.376153 1640.557 109.2736 Glass Fiber Filters -29.586 1.527355 867.4536 49.40557 Glass Fiber Filters -29.2294 2.269494 257.285 16.65542 Glass Fiber Filters -29.631 2.280269 640.671 40.68972 SPOM -29.7056 1.050393 756.2197 44.22241 17.10037 SPOM -29.1636 2.057357 307.5112 25.20909 12.19842 SPOM -29.7506 1.144351 1454.596 85.14077 17.0846 53

Wet Beech Leaves -31.5103 -0.06094 2287.308 82.33072 27.78195 Wet Beech Leaves -31.8448 -0.63822 3222.436 113.9689 28.2747 Wet Beech Leaves -31.8743 -0.54135 2558.566 91.03292 28.10594 Wet Beech Leaves -31.7097 0.095171 2782.505 99.45714 27.97692 Wet Beech Leaves -31.9298 -0.22618 2336.054 84.59847 27.61343 Dry Beech Leaves -32.1409 -0.70881 2593.101 96.45616 26.88373 Dry Beech Leaves -32.1257 -0.9094 1612.325 63.05475 25.57025 Dry Beech Leaves -32.4695 -0.67522 1970.082 60.03473 32.8157 Dry Beech Leaves -32.6957 -0.79578 2571.07 69.16162 37.17481 Dry Beech Leaves -32.2042 -0.83194 1785.591 64.94647 27.49326 Soil -29.1778 0.467811 132.0378 8.44632 15.63258 Soil -29.242 0.731283 221.8803 13.42504 16.52735 Soil -29.2792 0.616928 138.7733 9.078847 15.28534 Soil -29.2549 0.893041 208.2827 12.34827 16.86736 Soil -29.5329 0.691389 204.857 12.49458 16.39566 Caecidotea kenki -26.6953 4.390816 65.79932 12.2668 5.364018 Caecidotea kenki -26.2751 5.771786 161.2689 29.47618 5.47116 Caecidotea kenki -25.56 4.649543 644.787 137.4531 4.690959 Caecidotea kenki -25.5002 4.473961 1211.874 221.0049 5.483471 Caecidotea kenki -25.7025 4.339589 304.502 65.54108 4.645972 Crangonyx shoemakeri -25.7275 6.000828 447.021 95.06901 4.702069 Crangonyx shoemakeri -25.6998 6.078404 1174.858 271.7888 4.322687 Pseudolimnophila -27.2046 4.521053 2327.411 460.7191 5.051692 Aquatic Lumbricidae -27.3297 3.269048 2267.161 566.0292 4.005377 Aquatic Lumbricidae -27.1932 3.291554 1142.157 294.1689 3.882656 Aquatic Lumbricidae -27.1128 3.316275 1735.263 449.3861 3.861409 DOC -28.5978 0 DOC -28.6222 0

All of the isotope values for each sample are listed above for each study site and season. DOC = Dissolved organic carbon. SPOM = Suspended Organic Carbon.

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