2012 NORTHEASTERN NATURALIST 19(Monograph 9):1–42 The Fauna of Seepage Springs and Other Shallow Subterranean Habitats in the Mid-Atlantic Piedmont and Coastal Plain David C. Culver1,*, John R. Holsinger2, and Daniel J. Feller3 Abstract - A number of shallow groundwater habitats that occur in the Coastal Plain and Piedmont of the mid-Atlantic region are documented and described. These isolated tiny aquifers are underlain by clay (hypotelminorheic habitats) and exit at seepage springs, springs, tiled fields and tile drains, and shallow wells. These shallow groundwater habitats harbor species with reduced eyes and pigment that are limited to these habitats. The distribution of 23 such species—four planarians (Sphalloplana and Phagocata), one snail (Fontigens), 13 amphipods (Stygobromus), and five isopods (Caecidotea)—is documented, based on over 450 records. More species (16) were found in hypotelminorheic habitats than in other shallow groundwater habitats. Also, more species were found exclusively in either the Piedmont or Coastal Plain, but seven species were found along the boundary (Fall Line) between these physiographic provinces. Compared to surface waters in nearby habitats, hypotelminorheic water had higher conductivity, higher dissolved oxygen, and slightly lower pH. Introduction Most of the obligate inhabitants of caves have a characteristic, convergent morphology of reduced or absent eyes and pigment, and elongated, thin appendages (Culver and Pipan 2009). What is not so widely appreciated is that species with this convergent morphology, termed “troglomorphy” by Christiansen (1962), are often found in non-cave subterranean habitats. Among the most interesting and unusual of these are shallow groundwater habitats where troglomorphic animals live only a few centimeters beneath the surface. Although there were occasional earlier reports of troglomorphic species from these habitats in the mid-Atlantic Piedmont and Coastal Plain (e.g., Hubricht and Mackin 1940), it was not until the mid-1960s, due to the impetus of J.R. Holsinger, that these habitats began to be explored and sampled in a systematic manner. Rock Creek Park, in the heart of Washington, DC and administered by the National Park Service, was especially important in this regard. Close to the US National Museum of Natural History, troglomorphic flatworms, snails, isopods, and especially amphipods that were limited to these shallow subterranean habitats were collected and described. An astonishing total of five species of the amphipod genus Stygobromus was found 1Department of Environmental Science, American University, 4400 Massachusetts Avenue NW, Washington, DC 20016. 2Department of Biological Sciences, Old Dominion University, Norfolk, VA 23508. 3Maryland Department of Natural Resources, Wildlife and Heritage Service, Natural Heritage Program, c/o University of Maryland, Appalachian Laboratory, 301 Braddock Road, Frostburg, MD 21532. *Corresponding author - dculver@american.edu. 2 Northeastern Naturalist Vol. 19, Monograph 9 in Rock Creek Park (Culver and Šereg 2004, Pavek 2002), signaling that a rich, interesting fauna was present in these little-studied small habitats. With the exception of some artesian wells in the Edwards Aquifer of Texas (Holsinger and Longley 1980), no other area in the world has this many sympatric subterranean amphipod species. Because many of these species have very restricted ranges and were known from only a small number of sites, agencies charged with the monitoring and protection of at risk species, especially the Capital Region of the National Park Service (see Pavek 2002), the Maryland Natural Heritage Program, and the Virginia Natural Heritage Program, became interested in these habitats and their inhabitants, and supported inventory and taxonomic work. This effort resulted in a large increase in the number of records, additional new species, and more information about these types of habitats, which is summarized in this paper. This report has three major parts: (1) a description of the habitats, including their chemical and physical characteristics; (2) an annotated list of the obligate subterranean-dwelling species, together with locality records and distribution maps; and (3) some biogeographic and evolutionary considerations. Our geographic coverage is the Piedmont and Coastal Plain of Maryland, Virginia, and the District of Columbia (Fig. 1). Habitats Groundwater habitats come in a variety of forms, and the two best known ones are interstitial habitats (including the underflow of streams) with small habitat dimensions and caves with large habitat dimension (Botosaneanu 1986). Culver and Pipan (2008, 2009) identify a third major category—shallow subterranean habitats, which occur within a few meters of the ground surface and have variable habitat dimensions. Water emerges from these habitats in springs, which also take a wide variety of forms (Kresic 2010). Because springs provide access to these habitats, although indirectly, they are often the place where the groundwater fauna can be sampled. Historically, shallow wells (typically <15 m deep) also were places where the groundwater fauna could be sampled, but they have largely disappeared due to urbanization, population growth, and liability fears. The shallowest of these habitats, which are rather common, especially in some areas of the Coastal Plain and near the boundary (Fall Line) between the sediments of the Coastal Plain and the hard metamorphic rock of the Piedmont, are little more than wet spots. These habitats have been given a series of names, none of them entirely satisfactory, which has resulted in continuing confusion. Perhaps the earliest name used was “seep” (e.g., Holsinger 1967), but this term, in US usage at least, often refers to petroleum oozing out of the ground. Less confusing is the term “seepage spring”. According to Kresic (2010), a seepage spring is a diffuse discharge of water, where the flow cannot be immediately observed but the land surface is wet compared to the surrounding area. This description captures the essence of many of the mid-Atlantic seepage spring habitats—wet spots in the woods (Fig. 2). Kresic (2010) also provides a useful context for the classification of seepage springs within the general framework of springs. Flows 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 3 of seepage springs are typically less than 10 cm3 per second, making them eighthorder springs in Kresic’s extension of Meinzer’s (1923) categorization of springs by discharge. Seepage springs are gravity fed and situated in sediment. Kresic (2010) pointed out that variability of discharge is an important hydrological (and ecological) parameter, and indicated that if the ratio of the maximum to minimum discharge exceeds 10, then the spring can be considered highly variable. Because many seepage springs have little or no flow during hot, dry periods, they would be classified as highly variable. Figure 1. Map of the study area. Sampling sites with stygobionts are shown as gray dots, and the approximate location of the Fall Line is shown as a dotted line. 4 Northeastern Naturalist Vol. 19, Monograph 9 Seepage springs fit less comfortably into other spring classification schemes. Springer and Stevens (2009) defined 12 spheres of discharge, and seepage springs fall under the category of helocrene springs, i.e., springs that emerge with diffuse flow from low-gradient wetlands. However, more typical helocrene springs include soap holes or quicksand (Springer and Stevens 2009)! Seepage springs also have some characteristics of limnocrene springs (see also Danks and Williams 1991), ones that emerge into pools, but the fit to this classification is poor at best. Meštrov (1962) applied the term “hypotelminorheic” to shallow groundwater habitats that are vertically isolated from the water table and are “constituted of humid soils in the mountains, rich in organic matter and traversed by moving water” (authors’ translation). This groundwater habitat has usually been ignored in overall groundwater classification schemes (e.g., Hahn 2009). Juberthie (2000) included it in his discussion of subterranean habitats, but more as a special case than an intergral part of the subterranean realm. The very non-euphonious nature of the word hypotelminorheic (Greek roots expressed in French by a Croatian biologist) has even led to ridicule (Chapman 1993), but we believe the term is very useful. Based on Meštrov’s (1962) definition and his sketch of the habitat (redrawn as Fig. 3), Culver et al. (2006) proposed that the term “hypotelminorheic” be used to describe habitats with the following major features (see also Culver and Pipan 2008): Figure 2. Photograph of seepage spring in Scotts Run Regional Park, Fairfax County, VA. Photograph by W.K. Jones, used with permission. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 5 • A perched aquifer fed by subsurface water that creates a persistent wet spot. • Underlain by a clay or other impermeable layer typically 5 to 50 cm below the ground surface. • Rich in organic matter compared with other aquatic subterranean habitats. • Culver et al. (2006) also indicated that the drainage area of a seepage spring is typically less than 1 ha, that the seepage spring is in a shallow depression, and that the leaves are characteristically blackened and usually not skeletonized. Without a clay layer, water should tend to move vertically, and there would be no persistent water. The water exits at a seepage spring. The hypotelminorheic and seepage springs are the extreme end members of a series of groundwater habitats and their exits (springs in the broad sense) where troglomorphic species are found. At the other extreme are deep aquifers, such as the Edwards Aquifer in Texas, which harbors troglomorphic species hundreds of meters below the ground surface (Holsinger and Longley 1980). In the Piedmont, subterranean species could also occur in deep fractured rock aquifers and occasionally exit through springs. These fractured rock aquifers often extend relatively close to the surface, in saprolite in particular and regolith in general. Other shallow subterranean habitats, which do not comfortably fit the definition of the hypotelminorheic, occur close to the surface. For example, there are some seepage springs emanating from solid rock crevices with very thin soils on Bear Island in the lower Potomac River. In any event, our purpose here is not to redefi ne terms, but to catalog the fauna of shallow subterranean habitats and note their potential ecological and evolutionary importance. For most of the species discussed herein, it is groundwater and not the exit of the water to the surface, i.e., the groundwater/surface water ecotone (see Gibert 1991), that is their primary habitat. However, there are some species, such as the isopod Caecidotea kenki, that are likely concentrated around the ecotone itself (Fong and Kavanaugh 2010). The seepage spring is the point of collecting, but is not the shallow groundwater habitat itself. The habitat is clearly an example of a groundwater dependent ecosystem (Eamus and Froend 2006). It is also an Figure 3. Sketch of hypotelminorheic habitat in Medvenica Mountains, Croatia. Adapted from Meštrov (1962) by S. Gottstein. 6 Northeastern Naturalist Vol. 19, Monograph 9 isolated wetland, although a highly miniaturized one. Springs in karst areas may also harbor some of the shallow groundwater species discussed here, such as Caecidotea pricei in Washington County, MD. Although it is also found in drip pools and streams of caves, this species is more commonly collected in seepage springs and springs. While C. pricei is often restricted to the point of groundwater emergence, in larger springs it may inhabit extensive reaches of spring runs. Clay is a critical component of hypotelminorheic habitats, not only because it acts as a barrier to the downward movement of water, but also because during periods of drought, the water retained by the colloidal clay may serve as a refuge for invertebrates in the hypotelminorheic. Burrowing behavior in clay has been reported for two species of cave amphipods (Ginet and Decu 1977, Holsinger and Dickson 1977) collected from drip pools of water from epikarst that occasionally dry up, a habitat with some similarities to the hypotelminorheic (Culver and Pipan 2008). According to Ginet and Decu (1977), clay may also have some nutritional value. In the field, the provenance of either standing water or flowing water is not always evident. Small areas or flows of surface water may be a seepage spring or spring, or just standing water with no association with groundwater. Some sites appear to be seepage springs, but in reality they are merely temporary pools of rainwater. If seepage springs are defined by the presence of troglomorphic amphipods of the genus Stygobromus, there are very clear chemical differences between seepage springs, other small surface waters, and the underflow of nearby streams in George Washington Memorial Parkway in Fairfax County, VA (Table 1). Based on spring and summer measurements, conductivity and dissolved oxygen were higher in seepage springs, whereas pH and temperature were lower. These differences are sufficient to easily distinguish the three types of habitats. Higher conductivity is the result of longer residence times of water in the subsurface and the lower temperature is a reflection of the mean annual temperature in the region (about 14 °C). Not surprisingly, dissolved organic carbon concentrations (DOC) are much higher in seepage springs than in caves. DOC concentrations in epikarst drip water in caves average around 1 mg C L-1 (Simon et al. 2007) compared to 4.9 mg C L-1 for a seepage spring near Pimmitt Run in the George Washington Memorial Parkway (n = 7 samples, range = 1.6–9.2 mg C L-1; D. Fong, Department Table 1. Chemical parameters for the underflow of streams (hyporheic), seepage springs with the troglomorphic amphipod Stygobromus, and other small pools of water on the surface, presumably rainwater. Samples were taken in the George Washington Memorial Parkway, VA from March to September, 2003–2005. For details see Culver et al. (2006). Values are reported as mean ± S.E.(n). Hypotelminorheic Parameter Hyporheic (with Stygobromus) Other surface water Temperature (°C) 19.93 ± 0.55 (93) 11.77 ± 1.00 (16) 17.19 ± 1.58 (66) Conductivity (μS/cm) 309.01 ± 15.49 (68) 479.57 ± 102.81 (14) 281.62 ± 25.08 (37) Dissolved oxygen (mg/L) 5.53 ± 0.20 (93) 7.89 ± 0.75 (16) 5.81 ± 0.38 (65) pH 7.10 ± 0.07 (56) 6.25 ± 0.11 (16) 6.64 ± 0.07 (64) 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 7 of Biology, American University, Washington, DC, unpubl. data). Eight DOC measurements in seepage springs on Nanos Mountain in Slovenia yielded lower values (2.9 mg C L-1, range = 0.40 to 9.89 mg C L-1) that were still several times higher than cave waters (T. Pipan, Karst Research Institute at ZRC SAZU, Postojna, Slovenia, unpubl. data). Based on a ten-month monitoring period (March 2007 to January 2008) of the temperature of a seepage spring in Prince William Forest Park, Prince William County, VA, the habitat varies temporally (Fig. 4), although less so than in a stream 10 m away. From May to September, temperatures were depressed compared to the nearby surface stream, and approximated surface-water temperatures for the rest of the year. In spite of the variability, the amplitude of variation in seepage spring temperatures is less than that of surface waters. The maximum recorded temperature in the seepage spring was 22 °C compared to 28 °C in the nearby stream. The coefficient of variation of stream temperature for the data in Figure 4 was 49.8%, and the coefficient of variation of seepage spring temperature for the same period was 38.2%. This is a remarkable difference given the superficial nature of the hypotelminorheic and seepage spring habitats. In other areas where winters are more severe, seepage springs have higher winter temperatures as well as lower summer temperatures than surface water (Culver and Pipan 2008). Although not obvious in Figure 4, temperature in the seepage spring also varies daily, usually by less than 2 °C. Groundwater habitats in the study area are not limited to the hypotelminorheic and seepage springs. Tiled fields and associated tile drains (Fig. 5) are artificial habitats that mimic these habitats. Tiling is the laying of pipes at shallow depths (ca. 1.5–2 m) in fields to increase drainage. They are perhaps more common in the Figure 4. Hourly temperature from 7 April 2007 to 4 February 2008 in a hypotelminorheic habitat and adjoining stream in Prince William Forest Park, Prince William County, VA. Due to the scale, line thickness indicates the extent of daily fluctuations. 8 Northeastern Naturalist Vol. 19, Monograph 9 US Middle West, where troglomorphic species have also been found (Hubricht and Mackin 1940, Koenemann and Holsinger 2001). The tile drains often dry up during summer, but water persists in the pipes, which may act like clay in natural systems. In various places, especially near the headwaters of streams, small eighth-order springs (springs with discharges of less than 1 liter per second) can issue from naturally occurring tubes in sediments, up to several centimeters in diameter, a feature Kresic (2010) calls gushets, albeit miniature ones. They are often marshy, partly as the result of the lateral movement of water underground. Some of the records of species living in shallow subterranean habitats that we enumerate below are from stream headwaters. It is an interesting, very rarely studied habitat. There is no clear distinction between a small spring and a seepage spring, except perhaps for the volume of flow, and the nature of the opening. Certainly there are seventh- and sixth-order springs present (springs with discharges up to 1 liter per second). Many of these springs are exits for groundwater from fractured rock aquifers, and Kresic (2010) provides several examples and illustrations. Larger springs (fifth order and higher) are typically found in karst areas, geological features that are absent in the Coastal Plain and Piedmont except in extremely limited outcrops of soluble rock. In addition to a few larger springs in these outcrops, a few caves are also present. Rarely have stygobionts (obligate subterranean-dwelling aquatic species) been found in these caves, although Rust Cave in Loudoun County, VA is a notable exception. Finally, there are wells that intersect groundwater. All of the stygobiont records from wells that are known to us are from old wells (typically 5–10 m deep). Figure 5. Photograph of a tile drain with the tiled field in the background in Isle of Wight County, VA. Photograph taken in April 1983 by J.R. Holsinger, and originally published in Lewis and Holsinger (1985). 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 9 This range of shallow subterranean habitats, combined with the apparent ease with which species colonize drainage tiles and tile drain systems, suggests that there are even more such habitats, some of them perhaps little more than small cavities in water-logged soil. Tanja Pipan (pers. comm.) suggests that the phrase “aquatic edaphic” habitats may be a useful one to refer to the totality of these habitats. Geomorphology Throughout the mid-Atlantic region, physiography is strongly influenced by the underlying geology. Physiographic provinces within the study area are northeast-to-southwest-trending belts extending from New York to Georgia, with the Piedmont and Coastal Plain being the easternmost of these. The Piedmont comprises the western portion of the study area, including 30 km2 in Washington, DC, 6500 km2 in Maryland, and 17,300 km2 in Virginia. To the west it is mostly bounded by the Blue Ridge Mountains, an anticlinal ridge of metamorphic rock. The distinct topography of the Piedmont—rolling hills with deeply incised stream valleys—is comprised of two distinct geologic divisions, Late Proterozoic and Paleozoic igneous and metamorphic rocks with (Mesozoic) Triassic sedimentary rocks faulted into the metamorphic and igneous rocks (Fitcher and Baedke 2000). Areas underlain by sedimentary rock, primarily limestones, dolomites, shales, or sandstones are easily weathered to form lowlands, while plateaus, isolated mountains (monadnocks), and rolling hills are features associated with metamorphic or igneous rocks such as granite, gneiss, quartzite, phyllite, and gabbro (Swain et al. 2004). Rocks are strongly weathered in the Piedmont’s humid climate, and bedrock is generally buried under a thick (2–20 m) blanket of saprolite (Weeks 2001). Surface elevations in the Piedmont range from approximately 30 to 560 m asl. Conventional aquifers associated with this region are consolidated and vary by strata. Because of limited storage capacity, springs issuing from crystalline rock typically have low to moderate but highly variable flows, while springs flowing from limestone strata may flow in excess of 4000 L per minute (Otten and Hilleary 1985). The eastern-sloping rocks of the Piedmont extend beneath Coastal Plain sediments to form a basement layer. The surficial transition between the Piedmont and the Coastal Plain is the Fall Line, an irregular boundary often marked by waterfalls or rapids on streams. Small, isolated erosional remnants of Coastal Plain deposits are common west of the Fall line. At the Fall Line, precipitation typically infiltrates, flowing quickly within short groundwater flow paths to nearby streams in this area of highly permeable sands and gravels and significant relief (McFarland 1997). The Coastal Plain physiographic province encompasses 40 km2 of Washington, DC, 13,000 km2 of Maryland, and 34,000 km2 of Virginia. Generally, Coastal Plain sediments consist of an eastward-thickening wedge of unconsolidated, interbedded sands, shells, and clays, ranging in age from Early Cretaceous to Holocene (Meng and Harsh 1988). Sediment depths exceed 2 km along the Atlantic coast (Schmidt 1993). The Coastal Plain is often divided into two distinct 10 Northeastern Naturalist Vol. 19, Monograph 9 subprovinces based on topography and location; the westernmost portion, the Upper Coastal Plain, and to the east, the Lower Coastal Plain. The Upper Coastal Plain is higher in elevation, up to 105 m asl, and often has rolling hills and incised stream valleys. The deeply weathered deposits near Washington, DC and points north along the Fall Line (the Dissected Outcrop Belt) include some of the oldest landscapes in the mid-Atlantic Coastal Plain (Ator et al. 2005). The old age (5 million years) and composition of these sediments, as well as the complex geology of extensive faulting and fracturing, may be a factor in explaining the high groundwater faunal diversity in these areas. The Lower Coastal Plain lies east, bordering the Atlantic Ocean, and is easily identified by its flat, low-lying (<20 m asl) landscape that is dissected by the many tidal tributaries that drain into the Chesapeake Bay and coastal bays on the Atlantic coast. The Chesapeake Bay, a major feature of the Coastal Plain, was created about 5000 to 6000 years ago when the lower reach of the Susquehanna River in the Chesapeake lowland was flooded as meltwater from Pleistocene glaciers raised sea levels (Schmidt 1993). Shallow aquifers of the Coastal Plain are unconfined, though deeper aquifers are isolated by confining layers of primarily clay, some regional in size and extending under the Chesapeake Bay. Sampling and Collecting Collecting is primarily accomplished by hand collecting at seepage springs and tile drains. Because the hypotelminorheic habitat is so superficial, it is sometimes possible to sample an area of 10 m2 or more by systematically picking up leaves (Fig. 2). However, this is a potentially destructive form of sampling since it disrupts the structure of the habitat even if leaves are returned, and has been used only very occasionally (see Culver and Šereg 2004 for an example). In some cases, baiting has been performed using raw pieces of shrimp placed in a 500-ml plastic water bottle, which is cut in half and the top inverted into the bottom. This method sometimes yields numerous amphipods, isopods, and planarians, but, more frequently, the traps are found by Raccoons (Procyon lotor) and destroyed. The Bou-Rouch pump, widely used to sample the underflow of streams (see Bou and Rouch 1967, Culver and Pipan 2009), is ineffective because there is neither sufficient water nor coarse sediment in seepage springs. Regardless of which technique is used, some species are missed during each sampling event (so-called false negatives), thus necessitating repeated sampling. Shallow wells have usually been sampled by lowering a jar baited with raw shrimp, with holes punched in the lid, and left for 24 hours or more. Species Coverage Below we review current information about the systematics, distribution, and biology of four species of flatworms, one species of snail, thirteen species and one subspecies of amphipods, and five species of isopods. They comprise the known stygobiont fauna of the Coastal Plain and Piedmont regions of the 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 11 District of Columbia, Maryland, and Virginia. All of these species show some modification for subterranean life (troglomorphy), at least in terms of reduction of eyes and pigment. Most, especially the amphipods, have entirely lost eyes and pigment, and, at least at the gross morphological level, are indistinguishable from related cave-dwelling species (Culver et al. 2010). A few, however, retain considerable pigment and apparently functional eyes. The criterion for their inclusion was their restriction to subterranean habitats, not their morphology. Some additional species are often found in seepage springs, but they are more common in surface habitats. We have not included these species because their records in seepage springs are anecdotal and incomplete, and because they occur in other habitats. However, several amphipod and isopod species occur frequently enough that they may maintain permanent populations in some seepage springs and hypotelminorheic habitats, and are thus considered to be stygophiles. These species are the amphipods Crangonyx floridanus Bousfield, Crangonyx shoemakeri (Hubricht and Mackin), and Gammarus minus Say, and the isopod Caecidotea nodulus Williams. The planarian Phagocata morgani (Stevens and Boring) deserves special mention. It is highly variable in size (length 2–15 mm), and according to Kenk (1972), this species inhabits springs and cold creeks from New Brunswick, Canada, to North Carolina, and west to Wisconsin and Kentucky. Norden (1978) reported that 78 percent of the occurrences of this species in Maryland were at springs (presumably including seepage springs) and adjoining spring runs and brooks, and that populations farther downstream are usually not self-sustaining. Most of these occurrences were from the Piedmont (Norden et al. 1990). Kenk (1935) suggested that P. morgani cannot tolerate high summer temperatures and that it frequently occupies subterranean habitats, as do similar European species. It is also known from a few caves west of the study area (Norden 1978). While clearly not a stygobiont, P. morgani is nonetheless a frequent inhabitant of seepage springs, and probably the hypotelminorheic and other shallow aquatic subterranean habitats. What distinguishes it from the other stygophilic planarians is that it lacks pigment, although it does have eyes (Kenk 1972, Norden 1978). Why P. morgani is depigmented even though it occurs in many surface waters is unknown. Perhaps its original habitat was subterranean, maybe even hypotelminorheic, and it subsequently successfully colonized surface habitats. Another enigmatic species is the isopod Caecidotea hoffmani Lewis. It is depigmented and with vestigial eyes, suggesting that it lives in a shallow groundwater habitat. However, the only records are from a sphagnum bog and in the bryophyte Fontinalis near Suffolk, VA. The collections were made in the 1950s (Lewis 2009b), and no additional habitat information is available. There are almost certainly other stygobionts in the region. A number of tiny (<4 mm) amphipods of the genus Stygobromus have recently been discovered and described (Holsinger et al. 2011), and there are undoubtedly more to be collected and described. The collection and identification of flatworms essentially ceased 12 Northeastern Naturalist Vol. 19, Monograph 9 in the mid-1980s with the death of Roman Kenk, who collected and described many of the species. It is not a simple matter to preserve flatworms for taxonomic work. For example, Kenk (1977a) insisted on examining live specimens and then killed them in a nearly boiling solution of saturated HgCl2. Based on his work, it is clear that more species remain to be discovered and that the described species probably have wider distributions than are currently documented. We believe that Kenk’s work was sufficiently extensive to warrant inclusion in this paper. Finally, some groups have not been investigated at all and are likely to contain stygobionts, most especially copepods. Epikarst, another shallow subterranean habitat, contains many undescribed stygobiotic copepods (Pipan and Culver 2005), and seepage springs likely do also. For each species in the checklist, we give its type locality, a list of sites and a distribution map within the study area, a description of its biology, and localities where it has been found, if anywhere, outside of the study area. Collectively, more than 450 total records are included below. Unlike most caves and streams, seepage springs rarely have names, and thus it is difficult if not impossible to match old records with new ones. Seepage springs are often less than 100 m apart, making this problem especially difficult (prior to the advent of GPS technology). In old records, the type of habitat is often unclear. In addition, many sites that historically had interesting faunas are now destroyed because of the tremendous growth of the Baltimore– Washington metropolitan area. We have endeavored to include the full extent of all species’ ranges within the study area, but the large number of localities and ambiguities about matching old and new records makes the exact number of localities uncertain, especially for the most common species. As is apparent from the records, nearly all existing localities in the metropolitan area are in protected parks, especially those under the control of the National Park Service (e.g., Culver and Šereg 2004; Feller 1997a, b; Hobson 1997b, 1998; Hutchins and Culver 2008) and in military bases (e.g., Chazal and Hobson 2003; Hobson 1997a; Roble 1997, 2005). The existing sites are often extremely vulnerable to degradation. For example, seepage springs can be destroyed as a result of informal “social” trails where hikers are unaware that they are walking through these small wet habitats. For this reason, exact locations are not given, and distribution maps are shown by dots with a 5-km diameter that include the sites. More detailed site information is available in the original taxonomic descriptions, listed in the following sections, in reports to the National Park Service and US Fish and Wildlife Service (Culver and Šereg 2004; Feller 1997a, 1997b, 2005; Hutchins and Culver 2008), in reports by the Virginia Natural Heritage program (Chazal and Hobson 2003; Hobson 1997a, 1997b, 1998; Hobson and Roble 1998; Roble 1997, 2005; Roble and Derge 2001), and in conservation databases maintained by the Maryland and Virginia Natural Heritage Programs. Several museums house collections of the species discussed here, most notably the US National Museum of Natural History (Washington, DC) and the Virginia Museum of Natural History (Martinsville). 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 13 Annotated Checklist and Distribution Maps PHYLUM PLATYHELMINTHES Class Turbellaria Order Tricladida Family Kenkiidae Sphalloplana holsingeri Kenk 1977 Type Locality: shallow well (Biggers Spring; Fig. 6) on Edsall Road, Fairfax County, VA [now destroyed]. Other Records: known only from the type locality. Remarks: S. holsingeri is a blind, white species up to 15 mm in length and 1.5 mm in width when gliding. The type locality, a shallow well now destroyed and covered by urban development, was remarkably diverse, with S. subtilis (see below) and the amphipod Stygobromus tenuis potomacus (see Holsinger Figure 6. Photograph of Biggers Spring/Well near Edsall Road in Fairfax County, VA. The length of the line held by Roman Kenk (on the left) is the water depth. Person on the right is William Biggers. Photograph taken in March 1973 by J.R. Holsinger. For more details see Kenk (1977a). 14 Northeastern Naturalist Vol. 19, Monograph 9 1978). The water in the well was 1.8 m deep and reached to within 0.6 m of the casing enclosing the spring. Biggers Spring was enclosed in a brick structure with a removable concrete cover (Fig. 6). Considering the shallowness of the well, its position would seem to be that of the hypotelminorheic habitat, although the quantity of water present was greater than usually described for the hypotelminorheic. Sampling was performed using fresh shrimp bait. The distribution of S. holsingeri is shown in Figure 7. Sphalloplana hypogea Kenk 1984 Type Locality: two drain-tile outlets on a farm N of Chuckatuck, Isle of Wight County, VA. Other Records: known only from the type locality. Figure 7. Distribution of stygobiotic planarians found in the study area; Sphalloplana holsingeri and S. subtilis are endemic to the same locality, now destroyed. Gray dots represent all sampling sites with stygobionts. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 15 Remarks: S. hypogea is a blind, white species up to 17 mm in length and 3.0 mm in width when gliding. Drain-tiling of agricultural fields improves drainage and allows agricultural production in areas that were formerly wetlands. The tiles in this field are approximately 1.5 m beneath the surface. The drain-tile outlet is an artificial seepage spring. This habitat is common in the Midwest, and several stygobiotic species are known mostly from such sites, including the asellid isopod Caecidotea kendeighi (Forbes) and the amphipod Bactrurus mucronatus (Forbes) (Koenemann and Holsinger 2001, Lewis 2009a). The distribution of S. hypogea is shown in Figure 7. Sphalloplana subtilis Kenk 1977 Type Locality: shallow well (Biggers Spring) on Edsall Road, Fairfax County, VA [now destroyed]. Other Records: known only from the type locality. Remarks: S. subtilis is a very slender, blind, white species up to 16 mm in length and 1 mm in width when extended. It was found in the same locality as S. holsingeri, which has since been destroyed. See narrative of S. holsingeri for a description of the habitat. Sphalloplana subtilis was much less abundant than S. holsingeri (total of 10 versus 80 specimens collected, respectively). The distribution of S. subtilis is shown in Figure 7. Family Planariidae Phagocata virilis Kenk 1977 Type Locality: Seepage spring on the bank of the Patuxent River at McGruder Landing, Prince Georges County, MD. Other Records: MARYLAND: Calvert County: Seepage spring near Aquasio; Cecil County: Seepage spring at Elk Neck State Park; Prince Georges County: Seepage spring near Marlton; Queen Anne County: Seepage spring near Chestertown; Talbot County: Seepage spring near Choptank River. Remarks: P. virilis is a pigmented, eyed species up to 10 mm in length and 1.3 mm in width when extended (Kenk 1977b). Although it shows no obvious morphological modification for subterranean life, it has not been found in surface habitats. The type locality is unusual in being a seep only exposed at low tide (A. Norden, Maryland Department of Natural Resources, Annapolis, MD, pers. comm.). Additional collecting may change its status from stygobiont to stygophile. It is also known from a shallow groundwater site along the C&O [Chesapeake and Ohio] Canal National Historical Park in Washington County, MD (Norden et al. 1990). The distribution of P. virilis is shown in Figure 7. PHYLUM MOLLUSCA Class Gastropoda Order Mesogastropoda Family Hydrobiidae Fontigens bottimeri (Walker 1925) (Fig. 8) Type Locality: Glen Echo, Montgomery County, MD (precise location unknown, but a small seepage spring on the Dawson property in Glen Echo 16 Northeastern Naturalist Vol. 19, Monograph 9 may be the type locality inasmuch as it closely conforms to the description [Hershler et al. 1990]). Other Records: DISTRICT OF COLUMBIA: Seepage spring S of Military Road near Nature Center, Rock Creek Park (NPS); West Spring, Rock Creek Park (NPS); Wetzels Spring, Glover Archbold Park (NPS); two seepage springs near Reservoir Road, Glover Archbold Park (NPS). MARYLAND: Montgomery County: six seepage springs near Gold Mine Tract, C & O Canal National Historical Park (NPS); one seepage spring near Limekiln Branch, C & O Canal National Historical Park (NPS); four seepage springs near mouth of Cool Spring Branch, C & O Canal National Historical Park (NPS); two seepage springs in Chilton Woods, C & O Canal National Historical Park (NPS); one seepage spring in Seneca Creek State Park. VIRGINIA: Fairfax County: two seepage springs in Scotts Run Regional Park; three seepage springs in Great Falls Park (NPS); two seepage springs near Difficult Run, Great Falls Park (NPS); one seepage spring near Turkey Run, George Washington Memorial Parkway (NPS); Prince William County: seepage spring near Quantico Creek, Prince William Forest Park (NPS). Figure 8. Photograph of Fontigens bottimeri from a seepage spring in Scotts Run Regional Park, Fairfax County, VA. Snails are approximately 2 mm in length. Photograph by W.K. Jones, used with permission. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 17 Remarks: F. bottimeri is a small (1–3 mm) species with a highly variable morphology (Hershler et al. 1990). In addition to the localities listed above, it also occurs in a few caves in Maryland and Virginia, the westernmost of which is John Friend Cave in Garrett County, MD (Hershler et al. 1990). The body has pigment in some populations and lacks pigment in others, especially in caves west of the study area. Apparently, all individuals have eyes, but the bodies of relatively few specimens have been examined. Shell pigment is highly variable, even within a population (Fig. 8). Given its relatively large range for a subterranean species (Fig. 9), perhaps this taxon represents a complex of species. Some of the records Figure 9. Distribution of Fontigens bottimeri in the study area. Gray dots represent all sampling sites with stygobionts. 18 Northeastern Naturalist Vol. 19, Monograph 9 listed above are based on visual records of Fontigens not verified by dissection. Fontigens bottimeri is currently listed as an endangered species in Virginia by the Virginia Department of Game and Inland Fisheries. PHYLUM ARTHROPODA Class Crustacea Order Amphipoda Family Crangonyctidae Stygobromus araeus (Holsinger 1969) Type Locality: seepage spring N of Crittenden, formerly in Nansemond County, VA, now in the City of Suffolk. Other Records: VIRGINIA: Caroline County: seepage spring in ravine S of Bethel Church near junction of Routes 30 and 650; Chesapeake City: seepage spring S of South Norfolk; Isle of Wight County: two seepage springs SE of Bartlett; small stream S of Rescue; James City County: headwater tributary of Taskinas Creek, NE of Christensons Corner; four seepage springs in York River State Park; Mathews County: small spring on bank of Piankatank River at Twiggs Ferry; New Kent County: seep and small, spring-fed stream in Crump Swamp, E of Richmond; headwaters of Toe Ink Swamp near Quinton; Newport News City: eight seepage springs along Warwick River in Fort Eustis; Suffolk City (formerly Nansemond County): seepage spring W of Suffolk; Soren Spring SSW of Suffolk; seepage spring S of Crittenden; Surry County: seepage headwaters E of Highgate, Chippokes Plantation State Park; seven seepage springs in Chippokes Plantation State Park; York County: seepage spring at headwaters of tributary to Carter Creek, off Feeney Road in Camp Peary; small stream tributary and seepage spring of Skimino Creek in Camp Peary; four seepage springs in Colonial National Historical Park; seepage spring in Cheatham Annex. Remarks: S. araeus reaches a size of 5.5 mm (females) to 6.9 mm (males). Also found in a seepage spring just south of Virginia near Merchants Mill Pond in Gates County, NC, its range extends more than 120 km north to south and 70 km east to west. It occupies seepage springs, small springs, and seep-fed streams emerging from loosely consolidated Coastal Plain sediments ranging from upper Miocene to Pleistocene in age. All collections have been made between February and April, when flows are usually greatest and the animals move out of or are washed from the hypotelminorheic (Holsinger 1978). Some of the seepage springs where it was found were completely dry in the summer, and presumably the amphipods survived in the moist clay. The distribution of S. araeus is shown in Figure 10. Stygobromus caecilius Holsinger 2011 Type Locality: Belvedere seepage spring, Cecil County, MD. Other Records: known only from the type locality. Remarks: S. caecilius is a very small amphipod, reaching only 2.5 mm in body length. It is noteworthy in terms of the reduced number of spines and setae on most parts of the body, a characteristic of many interstitial Crustacea (Coineau 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 19 2000), but its habitat is a typical hypotelminorheic one. The seepage spring at the type locality is in relatively recent Coastal Plain sediments but is in contact with much older metamorphic rocks of Paleozoic age (Holsinger et al. 2011), and is threatened by proposed gravel mining. Stygobromus tenuis tenuis is also known from this site. The distribution of S. caecilius is shown in Figure 10. Stygobromus felleri Holsinger 2011 Type Locality: Funks Pond Spring, Cecil County, MD. Other Records: known only from the type locality. Remarks: S. felleri is a small amphipod, with males reaching a size of 4.5 mm. Funks Pond Spring is apparently developed in Paleozoic igneous rocks just west Figure 10. Distribution of Stygobromus araeus, S. caecilius, S. foliatus, and S. hayi in the study area. Gray dots represent all sampling sites with stygobionts. 20 Northeastern Naturalist Vol. 19, Monograph 9 of the eastern margin of the Piedmont physiographic province (Holsinger et al. 2011). Caecidotea pricei and Stygobromus pizzinii have also been collected from this site. The spring is located directly over a major thrust fault between gabbro and gneiss. The distribution of S. felleri is shown in Figure 11. Stygobromus foliatus Holsinger 2011 Type Locality: Spring in Nanjemoy Preserve (The Nature Conservancy [TNC)]), Charles County, MD. Other records: MARYLAND: Saint Marys County: two seepage springs near Poplar Hill Creek Spring. VIRGINIA: Caroline County: seepage spring and Figure 11. Distribution of Stygobromus felleri, S. indentatus, and S. kenki in the study area. Gray dots represent all sampling sites with stygobionts. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 21 pool, Pettigrew Wildlife Management Area; seepage spring and pond of Mount Creek, Fort A.P. Hill; King and Queen County: seepage spring near Exol Swamp; Westmoreland County: seepage spring at Voorhees Nature Preserve (TNC). Remarks: This is a relatively large species, with adults of both sexes reaching 8.0 mm. It is distinguished from all other Stygobromus by the presence of large, leaf-like sternal gills on pereonites 6 and 7 (Holsinger et al. 2011). Their function is unclear but could be a response to lowered oxygen availability. Stygobromus indentatus and a small-eyed species of the amphipod genus Crangonyx are also present at the type locality. The localities where S. foliatus is found are headwater seepage springs. All are in unconsolidated Coastal Plain sediments consisting primarily of sand, silt, clay, and gravels. Other stygobionts, including S. indentatus, Caecidotea jeffersoni, and an unidentified planarian, have been found at three of the localities. The distribution of S. foliatus is shown in Figure 10. Stygobromus hayi (Hubricht and Mackin 1940) Type Locality: small spring at south end of National Zoological Park, Washington, DC. Other Records: DISTRICT OF COLUMBIA: five seepage springs in Rock Creek Park (NPS); Rock Creek near Rapid Bridge in Rock Creek Park (NPS). Remarks: S. hayi reaches a size of 7.0 mm in females and 9.8 mm in males, slightly smaller than Stygobromus tenuis potomacus, with which it sometimes occurs. The distribution of S. hayi is highly restricted geographically, and the maximum linear extent of its range is 4 km. It has been on the US Federal Endangered Species List since 1982. Except for a single specimen found in extensive sampling of the sediments of Rock Creek, all specimens have been taken from seepage springs. This species appears in most seepage springs within its tiny range but what restricts it to this small area is unknown. Within its range, it can co-occur with S. kenki and S. sextarius, as well as S. tenuis potomacus. A hybrid population of S. hayi and S. tenuis potomacus has been reported from a spring between Suitland and Forestville in Prince Georges County, MD (Holsinger 1967). The distribution of S. hayi is shown in Figure 10. Stygobromus indentatus (Holsinger 1967) Type Locality: Tile drain outlet, 5 km NW of Suffolk, Nansemond County, VA [now City of Suffolk]. Other Records: MARYLAND: Anne Arundel County: Broad Creek Spring; shallow well at Smithsonian Environmental Research Center; Charles County: spring near Hancock Road, Nanjemoy Preserve (TNC); Prince Georges County: well near Aquaseo; Saint Marys County: seep near Poplar Hill Creek; three seepage springs near Chingville; Worcester County: Corbin Branch Spring; Cottingham Mill Run Spring. VIRGINIA: Caroline County: two seepage springs in Fort A.P. Hill; Fairfax County: seepage spring in Fort Belvoir; Isle of Wight County: seepage spring SE of Bartlett; seep near Cat Ponds, N of Chuckatuck; two drain tile outlets N of Chuckatuck; Lancaster County: Hand-dug well and seepage spring E of Whitestone; three seepage springs in Hickory Hollow Natural Area Preserve; 22 Northeastern Naturalist Vol. 19, Monograph 9 Northumberland County: two seepage pools in Hughlett Point Natural Area Preserve; two seepage springs in Bushmill Stream Natural Area Preserve near Howland; two seepage springs near Lewisetta; Suffolk City (formerly Nansemond County): outlets of drain tiles, ESE and NW of present business center of Suffolk; Westmoreland County: seepage spring in Voorhees Nature Preserve (TNC); two seepage springs in Westmoreland State Park. Remarks: S. indentatus is a relatively large species, with males reaching lengths of 9.7 mm and females 8.2 mm. It appears to be very closely related to S. pizzinii (Holsinger 1978). Stygobromus indentatus is also known from a shallow well in Nash County, NC, indicating that the species is not restricted to hypotelminorheic habitats. All of the habitats are in Coastal Plain sediments of Miocene age (Holsinger 1967). The distribution of S. indentatus is shown in Figure 11. A striking example of the value of persistence in collecting is the record from Fort Belvoir, where S. indentatus was found in only one of 134 seepage springs sampled (Chazal and Hobson 2003). Stygobromus kenki Holsinger 1978 Type Locality: seepage spring in Rock Creek Park, SE of Police Station (formerly North National Capital Parks headquarters) (NPS), Washington, DC. Other Records: DISTRICT OF COLUMBIA: two seepage springs in Rock Creek Park, in vicinity of Police Station; MARYLAND: Montgomery County: Burnt Mills seepage spring near Northwest Branch; Coquilin Run Spring. Remarks: S. kenki is a small species, with females reaching lengths of 5.5 mm and males 3.6 mm. The five known localities are all classic seepage springs, and the amphipods were found in wet leaf litter. Stygobromus tenuis potomacus is also present at all three District of Columbia sites, whereas S. sextarius occurs at two and S. hayi at one. Why S. kenki is often syntopic with other Stygobromus species is unknown. The report by Holsinger (1978) that S. kenki also occurs in Biggers Spring (well) in Fairfax County, VA was in error. That population represents an undescribed species (Holsinger 2009). The distribution of S. kenki is shown in Figure 11. Stygobromus obrutus Holsinger 1978 Type Locality: shallow well in woods, W of Danville, Pittsylvania County, VA. Other Records: known only from the type locality. Remarks: This is a small species with males reaching only 2.5 mm and females 3.6 mm in body length. Little is known about the type locality because the only collection was made in 1948, recent efforts to relocate the population were unsuccessful, and the original well is probably destroyed (Hobson and Roble 1998). It is either in early Paleozoic or Precambrian granite gneiss or Triassic sandstone. The presence of S. obrutus in a well suggests that it is found somewhat deeper than species inhabiting hypotelminorheic habitats. Its distribution (Fig. 12) is remarkable, being over 100 km distant from the nearest Stygobromus locality to the west in caves or to the east and north in shallow subterranean habitats. The distribution of S. obrutus is a cautionary tale about making broad generalizations about the distribution and history of the genus. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 23 Stygobromus paxillus Holsinger 2011 Type Locality: Prettyboy Dam spring, Baltimore County, MD. Other Records: known only from the type locality. Remarks: This is a small species, reaching a length of only 3.0 to 3.5 mm. All 20 specimens collected to date are females, suggesting that the species may be parthenogenetic (Holsinger et al. 2011). Culver and Holsinger (1969) reported other species of Stygobromus with either missing or rare males. The spring habitat of this species is apparently developed in sediments associated with Precambrian metamorphic rocks of the Piedmont; S. pizzinii has also been collected from the site. The distribution of S. paxillus is shown in Figure 13. Stygobromus phreaticus Holsinger 1978 Type Locality: well at Vienna, Fairfax County, VA. Other Records: VIRGINIA: City of Alexandria: well water; Fairfax County: seeping water along banks of deeply eroded stream in Fort Belvoir. Remarks: S. phreaticus is an intermediate-sized species with males reaching 6.8 mm and females 7.0 mm. This species is apparently not part of the hypotelminorheic fauna, as all collection sites are at least several meters beneath the ground surface. Together with Stygobromus obrutus, whose habitat is not well described, and the two planarian species of Sphalloplana found in shallow wells, they are the only species in this study that seem to be limited to these deeper habitats. The Fort Belvoir site is unusual in that the stream is deeply down cut as a result of stormwater Figure 12. Distribution of Stygobromus obrutus. Gray dots represent all sampling sites in the study area with stygobionts. Note its disjunct distribution relative to other species. 24 Northeastern Naturalist Vol. 19, Monograph 9 runoff (Chazal and Hobson 2003, Hobson 1997a). Although the water is seeping from its banks, it is not a seepage spring in the sense of Kresic (2010). Stygobromus phreaticus was only found in one of 44 survey sites in the stream ravine (Chazal and Hobson 2003). Most shallow, hand-dug wells, including the other two localities, have been destroyed by human activity, and we have no other way to sample this aquifer. The distribution of S. phreaticus is shown in Figure 13. Stygobromus pizzinii (Shoemaker 1938) Type Locality: Wetzels Spring, Glover Archbold Parkway (NPS), Washington, DC. Other Records: DISTRICT OF COLUMBIA: spring in Glover Archbold Parkway (NPS); MARYLAND: Baltimore County: two springs near Prettyboy Dam; Figure 13. Distribution of Stygobromus paxillus, S. phreaticus, and S. sextarius. Gray dots represent all sampling sites in the study area with stygobionts. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 25 Cecil County: Funks Pond Spring; Frederick County: artesian well on S side of Sugarloaf Mountain; seepage spring near mouth of Tuscarora Creek, C & O Canal National Historical Park (NPS); Howard County: Ellicott City (habitat not provided); Montgomery County: well on Mineshoe Island in Potomac River; four seepage springs near Cabin John Creek, C & O Canal National Historical Park (NPS); four seepage springs near mouth of Monocacy River, C & O Canal National Historical Park (NPS); seven seepage springs in Chilton Woods, C & O Canal National Historical Park (NPS); one seepage spring near Carderock, C & O Canal National Historical Park (NPS); two seepage springs near Gold Mine Tract, C & O Canal National Historical Park (NPS); four seepage springs near Edwards Ferry, C & O Canal National Historical Park (NPS); one seepage spring near Little Falls Dam, C & O Canal National Historical Park (NPS); four seepage springs near mouth of Cool Spring Branch, C & O Canal National Historical Park (NPS); one seepage spring near Swains Lock, C & O Canal National Historical Park (NPS); VIRGINIA: Arlington County: two seepage springs near Pimmits Run, George Washington Memorial Parkway (NPS); Fairfax County: seepage spring at Bullneck Run; seepage spring between Scotts Run and Bullneck Run; one seepage spring near Scotts Run, Riverbend Regional Park; two seepage springs near Gulf Branch, George Washington Memorial Parkway (NPS); six seepage springs near Turkey Run, Turkey Run Park, George Washington Memorial Parkway (NPS). Remarks: S. pizzinii is one of the largest species in the genus; males reach nearly 19 mm and females 16 mm. While specimens of this species are most commonly found in seepage springs and small springs, it has also been found in wells and rarely in caves. It is also known from Chester and Lancaster counties in Pennsylvania, and as far west as the Ridge and Valley physiographic province in Washington County, MD (Holsinger 1978). The most noteworthy cave occurrence is a large population of large individuals in an open lake in Reftons Cave in Pennsylvania, but this population may now be extinct (Holsinger 1967). The size range of adults is considerable, varying from 5.5 to 16 mm for females and 5.5 to 19 mm for males. The largest adults are recorded from Reftons Cave, whereas the smaller adults occur in seepage springs (with some exceptions). Stygobromus pizzinii can be quite common, but its appearance in any one site is rather unpredictable. Its distribution is shown in Figure 14. Stygobromus sextarius Holsinger 2009 Type Locality: seepage spring on southwest side of Beach Drive near Rock Creek, Montgomery County, MD. Other Records: DISTRICT OF COLUMBIA: walled seepage spring in National Zoological Park; two seepage springs near Sherrill Drive, Rock Creek Park (NPS); VIRGINIA: Arlington County: two seepage springs near Pimmitt Run, George Washington Memorial Parkway (NPS). Remarks: S. sextarius is a small species that only reaches a length of 3.5 mm in females and 2.5 mm in males. It has been collected from hypotelminorheic habitats, all within a distance of approximately 8 km of each other. The underlying bedrock of this area is a variable mixture of Paleozoic and Pre-Cambrian 26 Northeastern Naturalist Vol. 19, Monograph 9 schist, gneiss and quartz diorite, which occurs along the eastern margin of the Piedmont physiographic province. Stygobromus sextarius is often found syntopically with S. kenki and S. tenuis potomacus. Its distribution is shown in Figure 13. Stygobromus tenuis potomacus (Holsinger 1967) Type Locality: seep-fed bog in Burleith Woods, Glover Archbold Parkway (NPS), Washington, DC. Other Records: DISTRICT OF COLUMBIA: five seepage springs in vicinity of Police Station, Rock Creek Park (NPS); two seepage springs in vicinity of Walter Reed Hospital, Rock Creek Park (NPS); six seepage springs in National Capital Figure 14. Distribution of Stygobromus pizzinii in the study area. Gray dots represent all sampling sites with stygobionts. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 27 Park–East (NPS); stream source in Grover Archbold Parkway (NPS); seepage spring and bog in Burleith Woods (NPS); bog and brook near Chain Bridge; bog near Military Road; walled seepage spring at National Zoological Park; two seepage springs near Maryland state border, Rock Creek Park (NPS); spring near Dalecarlia Reservoir; three springs at Fort Stanton Park (NPS); MARYLAND: Anne Arundel County: sphagnum bog in Glen Burnie; spring near Bristol; Frederick County: artesian well near Sugarloaf Mountain; spring near top of Sugarloaf Mountain; spring near Jefferson; seepage spring near mouth of Tuscarora Creek, C & O Canal National Historical Park (NPS); seven springs along Furnace Branch; Harford County: seepage springs near Atkisson Reservoir; Howard County: seepage spring near Sucker Branch, Ellicott City; Montgomery County: spring near Batchellors Run Road; spring near Beach Drive; spring near Bell Run; three springs near Belle Cote Drive; seep near Berryville Road; bog and six springs at Burnt Hills; seep near Cape May Road; spring near Catalpa Court; nest of ants!, Fairland; Bear Island spring, C & O Canal National Historical Park (NPS); seepage spring in Chilton Woods, C & O Canal National Historical Park (NPS); spring near Countryside Drive; three seepage springs in Cropley Upland, C & O Canal National Historical Park (NPS); four seepage springs at Edwards Ferry, C & O Canal National Historical Park (NPS); three springs at Forest Glen Park; seven springs near Furnace Branch, C & O Canal National Historical Park (NPS); one seep at Germantown Bog; three seepage springs at Greenbelt Park; one seepage spring at Grist Mill Drive; three seepage springs at Gold Mine Tract, C & O Canal National Historical Park (NPS); two seepage springs at Cool Spring Branch, C & O Canal National Historical Park (NPS); two seepage springs at Limekiln Branch, C & O Canal National Historical Park (NPS); seepage spring near Northwest Branch; seepage spring near Park Vista Drive; seepage spring near Seneca, C & O Canal National Historical Park (NPS); two seepage springs near Swains Lock, C & O Canal National Historical Park (NPS); six seepage springs near Violets Lock, C & O Canal National Historical Park (NPS); one seepage spring near Watts Branch, C & O Canal National Historical Park (NPS); seepage spring at Wheaton Regional Park; seepage spring at Whitehaven; seepage spring at junction of Massachusetts and Wisconsin Avenues; Prince Georges County: bog in Cheverly; spring at University of Maryland; spring near Bristol; three seepage springs at Greenbelt Park; VIRGINIA: Alexandria City: seepage spring (unspecified locality); seepage spring near Beauregard Street; Arlington County: two seepage springs in Gulf Branch, George Washington Memorial Parkway (NPS); two seepage springs near Pimmitt Run, George Washington Memorial Parkway (NPS); seepage spring at Glencarlyn; well at Clarendon; well at Falls Church; Caroline County: two seepage springs near Gouldin, Fort A.P. Hill; seepage spring near Sales Corner, Fort A.P. Hill; Chesterfield County: seepage springs, pools, and a ditch off Courthouse Road opposite Pocahontas State Park; Fairfax County: four seepage springs in Fort Hunt Park, George Washington Memorial Parkway (NPS); three seepage springs near Dyke Marsh, George Washington Memorial Parkway (NPS); three seepage springs in Turkey Run Park, George Washington Memorial Parkway (NPS); two seepage springs near Difficult Run, Great Falls Park (NPS); 28 Northeastern Naturalist Vol. 19, Monograph 9 four seepage springs near Great Falls, Great Falls Park (NPS); two seepage springs in Wolf Trap Park for the Performing Arts (NPS); two seepage springs in Scotts Run Regional Park; pool at Dyke; pools between Belle Haven and Dyke; unspecifi ed habitat near Mt. Vernon; seepage springs, spring runs, and bog near Scotts Run; seepage spring in Lake Accotink Park, Springfield; shallow well and seepage spring off Edsall Road, Springfield; seepage spring ESE of Fairfax; pond in Mc- Clean; at least 60 seepage springs in Fort Belvoir; 10 seepage springs in Pohick Bay Regional Park; two seepage springs in Northern Virginia Regional Park; one seepage spring in Gunston Hall Plantation; one seepage spring in Mason Neck State Park; Fauquier County: spring on Bull Run Mountain; James City County: seepage spring in York River State Park; Loudoun County: stream and spring bog near Middleburg; Prince William County: two seepage springs in Prince William Forest Park (NPS); three seepage springs in Manassas National Battlefield Park (NPS); well (no further details available). Remarks: S. tenuis potomacus, shown in Figure 15, is a large species, with males reaching 16.5 mm and females 9.0 mm. It is nearly ubiquitous in seepage springs in the lower Potomac River drainage (Fig. 16), as the frequency of records indicates. Given the ambiguity in names of seepage springs and springs, it is difficult to know exactly how many populations have been found, but the total is Figure 15. Photograph of Stygobromus tenuis potomacus from a seepage spring in Scotts Run Regional Park, Fairfax County, VA. Amphipod is approximately 1 cm in length, with the head to the left. Photograph by W.K. Jones, used with permission. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 29 over 200. Along with Caecidotea kenki, S. tenuis potomacus is almost diagnostic of shallow groundwater habitats within its range. This amphipod occurs in the interstices of unconsolidated sands, gravels, and silts of the Coastal Plain and in crevices and joints of crystalline rocks of the Piedmont (Holsinger 1967). It has also been reported from a well somewhere near Richmond but not enough information is available even to place it in a county with certainty (Holsinger 1967). Egg production in females averaged 6.9 (S.D. = 1.1, n = 24) and occurred from March to June (Holsinger 1967). Culver and Poulson (1971) reported a standard metabolic rate of 2.1 microliter/mg/hr. There is no morphological difference between populations of S. tenuis potomacus on opposite sides of the Potomac River, which is not surprising because this amphipod is occasionally found in Figure 16. Distribution of the subspecies of Stygobromus tenuis in the study area. Gray dots represent all sampling sites with stygobionts. 30 Northeastern Naturalist Vol. 19, Monograph 9 hyporheic habitats, which extend across the river. There are additional records from seepage springs in Jefferson County, WV, and the Adams-Franklin county line in Pennsylvania (Holsinger 1978). While S. tenuis potomacus can be found throughout the year, it is most common in spring, when seeps are flowing more strongly. Fong and Kavanaugh (2010) reported that its abundance at one seepage spring along Pimmitt Run in Arlington County, VA, dramatically decreased when water temperatures exceeded 14 °C. The record from an ant nest is very strange, and we have no further details or explanation. The distribution of S. tenuis potomacus is shown in Figure 16. Stygobromus tenuis tenuis (Smith 1874) Type Locality: wells at Middletown, CT. Other Records: MARYLAND: Anne Arundel County: headwaters of Chase Creek; Jabez Rocks spring; Baltimore County: spring-fed stream in Phoenix; two springs at Prettyboy Reservoir; Calvert County: spring near Calvert Cliffs; Carroll County: two seepage springs in Alesias Swamp Woods; Cecil County: four springs in Belvedere Woods; Funks Pond Spring; Dorchester County: ditch near Cambridge; Harford County: seepage spring near Sandy Hook Road; seepage spring near Stafford Road; seepage spring at Susquehanna State Park; Prince Georges County: Marvin Seger Farm spring; Queen Annes County: two springs near Wye Mills; Talbot County: well near Trappe; VIRGINIA: Northampton County: from pitfall traps near interdunal pond in Savage Neck Natural Area Preserve near Eastville. Remarks: S. tenuis tenuis reaches a length of 12.0 mm in males and 9.7 mm in females. It occurs from southern New England south to New York City, and then again in eastern Maryland, including the Delmarva Peninsula (Holsinger 1978). In the study area, it is found to the north of the Potomac River drainage (Fig. 16). The Virginia record is remarkable both for its location near the tip of the Delmarva Peninsula and for the unusual habitat. Several specimens were captured in flooded terrestrial pitfall traps near an interdunal pond in a sandy area (S. Roble, Virginia Department of Conservation and Recreation, Division of Natural Heritage, Richmond, VA, pers. comm.)! Order Isopoda Family Asellidae Caecidotea jeffersoni Lewis 2009 Type Locality: seepage spring in Voorhees Nature Preserve (TNC), Westmoreland County, VA. Other Records: known only from the type locality. Remarks: C. jeffersoni is a medium-sized isopod, reaching 8.0 mm in males and 7.6 mm in females. Lewis (2009b) reported three similar populations from seepage springs in James City and King William counties, VA and Virginia Beach City, but those specimens are larger and possess tiny eyes. Further analysis is needed before their specific status can be determined. The distribution of C. jeffersoni is shown in Figure 17. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 31 Caecidotea kenki (Bowman 1967) Type Locality: spring SW of Nature Center, Rock Creek Park, Washington, DC. Other Records: DISTRICT OF COLUMBIA: six seepage springs near Sherrill Drive and Police Station, Rock Creek Park (NPS); Wetzels Spring and two seepage springs, Glover Archbold Park (NPS); small stream just off Blagden Avenue; MARYLAND: Montgomery County: four seepage springs near Little Falls Dam, C & O Canal National Historical Park (NPS); seepage spring at Carderock, C & O Canal National Historical Park (NPS); seepage spring at Glen Echo; seepage spring at Cabin John; seepage spring and stream at Kensington; Prince Georges County: stream flowing into Sligo Branch; VIRGINIA: Arlington County: two Figure 17. Distribution of Caecidotea jeffersoni and C. kenki in the study area. Gray dots represent all sampling sites with stygobionts. 32 Northeastern Naturalist Vol. 19, Monograph 9 seeps near Pimmit Run; spring at Glencarlyn; Fairfax County: ten seepage springs near Turkey Run, George Washington Memorial Parkway (NPS); one seepage spring near Gulf Run, George Washington Memorial Parkway (NPS): two seepage springs near Difficult Run, Great Falls Park (NPS); one seepage spring on CIA Headquarters grounds; four seepage springs in Scotts Run Regional Park; one seepage spring in Wolf Trap Park for the Performing Arts (NPS); stream near Bull Neck Run; Prince William County: one seepage spring in Manassas National Battlefield Park (NPS); one seepage spring in Prince William Forest Park (NPS). Remarks: This is a highly variable species morphologically. Males can reach 14 mm in body length but are typically much smaller. Ovigerous females are usually 7 to 8 mm (Bowman 1967). The eyes are small, and pigment is variable but reduced relative to surface-dwelling species of Caecidotea (Fig. 18). Within its range, C. kenki is nearly ubiquitous in seepage springs. Fong and Kavanaugh (2010) found this species in a seepage spring during all months of the year and at all temperatures. Caecidotea kenki is likely much more common than the above records indicate. It is also reported from two caves in Indiana and Fayette counties, PA (Bowman 1967), as well as a spring along the Appalachian Trail in Fauquier County, VA, just outside of the study area. It has not been sampled as thoroughly as amphipods in the genus Stygobromus. The distribution of C. kenki is shown in Figure 17. Figure 18. Photograph of Caecidotea kenki (head facing to the right) from a seepage spring in Scotts Run Regional Park, Fairfax County, VA. Isopod is approximately 8 mm in length. Photograph by W.K. Jones, used with permission. 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 33 Caecidotea phreatica Lewis & Holsinger 1985 Type Locality: Seepage spring near Murphy’s Pond, NW of Suffolk (formerly Nansemond County, now City of Suffolk), VA Other Records: VIRGINIA: Isle of Wight County: tile drain on farm N of Chuckatuck; City of Suffolk (formerly Nansemond County): three tile drains N of Chuckatuck; two shallow wells N of Chuckatuck; seep-fed pool NW of Suffolk. Remarks: The collection sites, all apparently hypotelminorheic habitats, occur in unconsolidated silt, sand, and clay sediments of Pleistocene age. The appearance of this species in drain tile outlets is seasonal, suggesting that it is periodically Figure 19. Distribution of Caecidotea phreatica, C. pricei, and C. vandeli in the study area. Gray dots represent all sampling sites with stygobionts. 34 Northeastern Naturalist Vol. 19, Monograph 9 washed out of its subterranean habitat. The distribution of C. phreatica is shown in Figure 19. Caecidotea pricei Levi 1949 Type Locality: Refton Cave, Lancaster County, PA. Other Records: MARYLAND: Cecil County: Funks Pond Spring; Montgomery County: three springs in Chilton Woods; VIRGINIA: Loudoun County: Rust Cave. Remarks: This species frequently inhabits caves in Maryland, Pennsylvania, Virginia, and West Virginia (Fong et al. 2007, Holsinger and Culver 1988, Holsinger and Steeves 1971, Lewis et al. 2011). Rust Cave is a very shallow cave developed in a limestone conglomerate. Caecidotea pricei has also been reported from springs in these karst areas, with most locality records from this habitat. Its distribution in the study area is shown in Figure 19. Caecidotea vandeli (Bresson 1955) Type Locality: Erhart Cave, Montgomery County, VA [now destroyed]. Other Records: MARYLAND: Frederick County: Gum Spring in Brunswick Town Park; Montgomery County: seepage spring near mouth of Goose Creek, C & O Canal National Historical Park (NPS); two springs in Three Spring Hollow, C & O Canal National Historical Park (NPS), spring at Edwards Ferry, C & O Canal National Historical Park (NPS); spring at Seneca State Park. Remarks: This species is found in many caves in the Valley and Ridge Province of Virginia (Holsinger and Culver 1988) and likely dispersed into the Piedmont from this area. Its distribution is shown in Figure 19. Discussion An ecological hypothesis Table 2 lists the shallow subterranean habitats (seepage springs, springs, tile drains, and shallow wells) in which the stygobiotic species in the study area have been found. The most common habitat was seepage springs, followed by other springs. Three species were found in tile drains, including Sphalloplana hypogea, which was found nowhere else. Only four of the 19 species and subspecies have been found in caves, with all but one of these records originating from outside of the study area. In this study, shallow subterranean habitats were the predominant habitat for stygobionts. According to the terminology we use in this monograph, the hypotelminorheic (shallow aquatic subterranean habitats) emerges at seepage springs. For most of the year when the seepage springs are flowing, they drain into streams and rivers. This presents three more or less distinct habitats, as do springs in general: The hypotelminorheic, underlain by clay and in constant darkness, The seepage spring, an ecotone between the groundwater of the hypotelminorheic and the spring run and stream, and The stream or river that drains the hypotelminorheic and seepage spring. The species found in springs and seepage springs can be tentatively assigned to these different habitats. All of the Stygobromus species, except S. phreaticus, S. obrutus, and perhaps S. felleri, are likely hypotelminorheic species at least 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 35 in part. Stygobromus phreaticus and S. obrutus seem to be part of a deeper groundwater community, as do the planarians Sphalloplana holsingeri and S. subtilis. This deeper, phreatic community is very poorly sampled primarily because wells, especially shallow wells, have largely disappeared from the landscape. In addition to the remaining Stygobromus species, the isopod Caecidotea jeffersoni and the planarian Sphalloplana hypogea are most likely hypotelminorheic species. Based on studies of Stygobromus tenuis potomacus, S. kenki, and S. hayi (Culver and Šereg 2004, Fong and Kavanaugh 2010), the appearance of hypotelminorheic species in seepage springs is seasonal, and they are most common at times of high flow, or relatively cool temperatures, or both. Although it has not been demonstrated for any of these species, we suspect that they survive dry periods in the underlying clay layer, or possibly deeper in fractured rock aquifers in the Piedmont. Table 2. Habitats from which each mid-Atlantic stygobiotic species has been reported. Cave habitats are typically not in the study area. Species Seepage springs Springs Tile drains Shallow wells Caves Turbellaria: Tricladida Sphalloplana holsingeri X Sphalloplana hypogea X Sphalloplana subtilis X Phagocata virilis X Gastropoda: Mesogastropoda Fontigens bottimeri X X X Crustacea: Amphipoda Stygobromus araeus X X Stygobromus caecilius X Stygobromus felleri X Stygobromus foliatus X X Stygobromus hayi X Stygobromus indentatus X X X X Stygobromus kenki X Stygobromus obrutus X Stygobromus paxillus X Stygobromus phreaticus1 X Stygobromus pizzinii X X X X Stygobromus sextarius X Stygobromus tenuis potomacus X X X Stygobromus tenuis tenuis2 X X X Crustacea: Isopoda Caecidotea jeffersoni X Caecidotea kenki X X Caecidotea phreatica X X X Caecidotea pricei X X Caecidotea vandeli X X X Total 16 12 3 9 4 1S. phreaticus has been found in seeping water along a stream bank, but not a seepage spring. 2S. tenuis tenuis has also been found in an interdunal pond. 36 Northeastern Naturalist Vol. 19, Monograph 9 Three species—the isopod Caecidotea kenki, the snail Fontigens bottimeri, and the planarian Phagocata morgani—are primarily denizens of seepage springs. Compared to the hypotelminorheic fauna, these species are more or less present year-round, and morphologically variable. They all exhibit some eye and pigment reduction as well as variability within and between populations. In the Potomac River basin, the presence of C. kenki and the hypotelminorheic specialist Stygobromus tenuis potomacus are nearly infallible indicators of the presence of hypotelminorheic habitats in the Coastal Plain. In the Piedmont, F. bottimeri is very abundant at the emergence of permanently flowing shale springs, but it is absent or difficult to find during the summer when flow rates decline. It is likely that most of the population resides underground in the rock fracture aquifer. Caecidotea kenki occurs in all types of springs in the Potomac River basin in the west, including karst, shale, and sandstone seeps through high volume springs. Finally, some of the species found in streams—including the amphipods Crangonyx floridanus, C. shoemakeri, and Gammarus minus and the isopod Caecidotea nodulus—are occasionally found in seepage springs (Hutchins and Culver 2008). Conversely, the seepage spring specialists are sometimes found in streams. This is especially true of Phagocata morgani, and Norden (1978) suggests that the numerous records of this species from headwater streams represent sink, rather than source, populations. An evolutionary hypothesis At least four major invertebrate groups (planarians, molluscs, amphipods, and isopods) have invaded aquatic shallow subterranean habitats, and it is quite possible that at least amphipods have invaded these habitats several times (Culver et al. 2010, Holsinger 2005). Since no molecular phylogeny or cladistic analysis exists for any of the species found in these habitats, discussion about their evolutionary history must be highly speculative. One of the ongoing debates about subterranean biogeography (Culver and Pipan 2009) is whether colonization is active (adaptive shift hypothesis) or passive (climate relict hypothesis). Given that hypotelminorheic habitats, and more particularly the underlying clay layer, would be a refugium for aquatic species during droughts, it is tempting to suggest that passive stranding in these habitats occurred, but the distinction between active and passive colonization is really quite small in this context. A more interesting, or at least a potentially more tractable question, is the role of dispersal relative to vicariance in both speciation and the overall distribution of aquatic shallow subterranean species (Holsinger 2005). The occurrence of some species in the study area, such as Caecidotea pricei and C. vandeli, almost certainly resulted from dispersal from cave regions located to the west, where the bulk of their populations are found. Several species, especially Fontigens bottimeri, have larger ranges (>100 km maximum linear extent), suggesting that they may represent a complex of cryptic species. However, most of the stygobionts included in this study are either limited to the study area or have few populations outside of it. The genus Stygobromus 2012 D.C. Culver, J.R. Holsinger, and D.J. Feller 37 is especially interesting: of the fourteen species and subspecies found in the study area, three (S. araeus, S. pizzinii, and S. indentatus) have been assigned to species groups (see Culver et al. 2010, Holsinger 1978) that are exclusively found in shallow subterranean habitats. It is difficult to imagine how their ancestors could be cave-dwelling species in other regions; rather, it seems more likely that they were surface-dwellers or perhaps species that inhabited the underflow of streams and rivers (hyporheic) in the Coastal Plain and Piedmont. The hypothesis that their ancestors were hyporheic species is especially attractive because of the similarities of hyporheic and hypotelminorheic habitats (Culver and Pipan 2008). However, no hyporheic species or populations of Stygobromus have been found in the region. Extensive sampling of the hyporheic of the Potomac River and several tributaries using Bou-Rouch pumps has yielded only a few scattered specimens of Stygobromus with densities less than 1 per 100 L (Culver and Šereg 2004, Hutchins and Culver 2008). Other Stygobromus species may have arisen from deep subterranean members of their same species group (e.g., S. obrutus and S. kenki [Holsinger 1978]), but it is still difficult to account for potential dispersal distances of up to 100 km. Most species in our study area were distributed in either the Coastal Plain or Piedmont, but not both. The only exceptions were the two subspecies of Stygobromus tenuis. The distribution of S. tenuis potomacus is especially interesting because it includes the Piedmont and sites along the Fall Line (Fig. 16). The Fall Line may be a dispersal corridor, as is suggested by the distributions of Stygobromus caecilius, S. hayi, S. kenki, and S. phreaticus, all of which range along the Fall Line. Conservation and protection Hand-dug wells, which were widespread during Colonial times in the mid-Atlantic region, are now mostly distant memories. They were generally considered to be physical risks that contained water unsafe to drink, and were typically filled in. The wells themselves were not the subterranean aquatic habitat, but provided human access to it. The current status of species like Sphalloplana holsingeri and S. subtilis cannot be determined (both are possibly extinct), but the intense urbanization of their known ranges does not lead us to any optimism. Seepage springs face many of the same risks as hand-dug shallow wells. They are often perceived as being little more than annoying areas of poor drainage, and thus are especially vulnerable to draining, filling, and/or contamination. Fortunately, personnel of many of the region’s parks, especially the national parks, are becoming aware of these habitats and their faunas (e.g., Pavek 2002). Seepage springs are increasingly recognized as important habitats, and their protection is becoming part of park planning. Acknowledgments D.C. Culver was supported by grants from the National Capital Region of the National Park Service. Drs. Daniel W. Fong and Tanja Pipan reviewed earlier versions of the manuscript and made many helpful suggestions. Justin Shafer of Old Dominion 38 Northeastern Naturalist Vol. 19, Monograph 9 University assisted in plotting site locations in southeastern Virginia. We thank the staff of the following National Parks for their help in collecting and locating sites: C & O Canal National Historical Park, George Washington Memorial Parkway, Manassas National Battlefield Park, National Capital East, Prince William Forest Park, Rock Creek Park, and Wolf Trap Park for the Performing Arts. Critical to this study were the many surveys of amphipods in eastern Virginia by the staff of the Virginia Department of Conservation and Recreation Natural Heritage Program, especially Steven Roble and Christopher Hobson. D.C. Culver thanks Dr. Florian Malard for introducing him to sampling techniques for seepage springs and to Dr. Diane Pavek for encouraging studies in the parks of the National Capital Region (NPS). D.J. Feller thanks all of the Maryland DNR staff that helped with field work. Taxonomic study and description of the four recently described species of Stygobromus in the study area was supported in part by State Wildlife Grant funds provided to the state wildlife agencies by US Congress and administered through the Maryland Department of Natural Resources’ Wildlife and Heritage Service. D.J. Feller was supported by grants from the National Capital Region of the NPS, by State Wildlife Grant funds provided to the Maryland Department of Natural Resources’ Wildlife and Heritage Service, the US Fish and Wildlife Service, and State of Maryland Endangered Species and Chesapeake Bay Tax Check Off. Publication costs were supported by a grant from the Cave Conservancy of the Virginias, as part of its continuing support of the analysis of the subterranean fauna of Virginia. Literature Cited Ator, S., J. Denver, W. Krantz, W. Newell, and S. Martucci. 2005. A surficial hydrogeologic framework for the mid-Atlantic Coastal Plain. 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