2013 NORTHEASTERN NATURALIST 20(1):185–196 The Second Northernmost Cave-adapted Fish in the World? Groundwork on the Tytoona Cave Sculpin Population Luis Espinasa1,*, Alexandra Mendyk1, Emily Schaffer1, and Amy Cahill1 Abstract - A new cave population of sculpin fish from Central Pennsylvania is described that, if confirmed to be cave adapted, would become the second northernmost caveadapted fish in the world. The Tytoona Cave fish lack the suite of modifications typical of troglomorphic populations. Their eyes, pectoral fins, and mouths appear to be as large as those of their surface counterparts, they have the same number of cephalic lateralis pores, and their pigmentation levels do not appear to be much lower. Nonetheless, they are considered to be cave adapted due to the presence of ovigerous females, a lack of evidence for starvation, and primarily because the cephalic lateralis pores (part of the lateral line system) are significantly larger than those of similar-sized surface fish. It may be that the Tytoona Cave population only has some emergent cave adaptations because in high latitudes the extent of polar ice sheet migration during the Pleistocene era restricted colonization by fish at least until 12 ka ago, when the ice age ended. Introduction Since the description of the first cave-adapted fish, Amblyopsis spelaea (De Kay 1842), over 100 species of fish with troglomorphic traits (e.g., reduction in pigmentation and eyes) have been found in caves and other subterranean habitats around the world (Burr et al. 2001; Espinasa et al. 2001; Proudlove 2006, 2010; Romero and Paulson 2001). In the development of modern biology as a science, cavefish have played a significant role. For example, in the relatively new field of evolutionary developmental biology (Evo-Devo), cavefish have been viewed as a model system since they have revealed some of the molecular and cellular mechanisms involved in trait modification, the number and identity of the underlying genes and mutations, the molecular basis of parallel evolution, and the evolutionary forces driving adaptation to the cave environment (Jeffery 2001, 2009, 2010). Contrary to popular belief, the importance of cavefish and other cave animals does not rest solely on their degenerate depigmentation or eyeless attributes. Cavefish have evolved constructive adaptations as well, such as specialized appendages, longer life spans, hypersensitive olfactory systems, and revamped gustatory and mechanosensory systems (Culver and Pipan 2009, Poulson and White 1969). In high latitudes, troglomorphic fish are absent despite the presence of caves and other suitable subterranean habitat (Romero and Paulson 2001, Proudlove 2006). One factor that likely constrains the distribution of cavefishes is the extent of polar ice sheet migration during the glacial periods of the Pleistocene Epoch. At the peak of the last Wisconsinan Period ≈20 ka ago, ice sheets covered most 1School of Science, Marist College, 3399 North Road, Poughkeepsie, NY 12601. *Corresponding author - luis.espinasa@marist.edu. 186 Northeastern Naturalist Vol. 20, No. 1 of the Northern Hemisphere above 40–50°N (Flint 1971). Therefore, most northern caves were not available for colonization by fish until 12 ka ago, when the Wisconsinan Period ended. Espinasa and Jeffery (2003) described the northernmost cave-adapted fish in the world (41°9'N). This population of sculpin (Cottidae: Scorpaeniformes: Actinopterygii) inhabits Eiswert #1 Cave (Stone 1953) in the Nippenose Valley, Lycoming County, in Central Pennsylvania and was assigned to the Cottus bairdi-cognatus complex (Espinasa and Jeffery 2003). Individuals of this cave population retain, although reduced, functional eyes and some pigmentation. They are morphologically distinct from surface sculpin of nearby streams by their wider and more abundant mandibular pores (Fig. 1), wider heads, longer pectoral fins, Figure 1. Lateral views of heads. A) Pennsylvania Grotto Sculpin from the Nippenose Valley. B) Surface fish from the Nippenose Valley. Arrows point to mandibular pore VI (below) and the extra pore (above) found in troglomorphic specimens in the Nippenose Caves, but absent in those from Tytoona Cave. Size differences in the pores between A and B are similar to the differences found between Tytoona Cave specimens and surface fish. Tytoona Cave specimens have large eyes as in B. Scale bar: 0.5 cm. (Photo from Espinasa and Jeffery 2003). 2013 L. Espinasa, A. Mendyk, E. Schaffer, and A. Cahill 187 and reduced tectum opticum (area of the brain dedicated to vision). The common name by which they are now known is the Pennsylvanian Grotto Sculpin. Eiswert #1 Cave is part of the West Branch of the Susquehanna River drainage. Because of the occurrence of large karst areas with caves along its West Branch and the presence of the assumed ancestral species of the Pennsylvanian Grotto Sculpin (Espinasa and Jeffery 2003), the Cottus bairdi Girard (Mottled Sculpin) and/or the Cottus cognatus Richardson (Slimy Sculpin), within the drainage, it is possible that other populations of sculpins may have successfully invaded the cave environment. After interviewing several members of the local caver community, it was revealed that there were sightings of sculpin within the subterranean stream of Tytoona Cave, near the town of Altoona, Blair County, also in Central Pennsylvania (Fig. 2). The objective of this study is to assess if the Tytoona sculpin population is cave-adapted, which would make it the second cave-adapted population in the world north of 40°N. Methods Field-site description Tytoona Cave Nature Preserve is located in Sinking Valley, Blair County, PN, (40°36'04"N, 78°13'01"W., 274 m.a.s.l.; Fig. 2) between the cities of Tyrone and Altoona (hence the name). This preserve is owned by the National Speleological Society. The large 10- x 5-m cave entrance (Fig. 3) is located in the bottom of a large wooded sinkhole with a running stream. The surrounding area has no surface streams. A stream connection to nearby Arch Spring (Fig. 4) has been identified by dye tracing, and is 1.22 km straight-line distance from the main cave Figure 2. Map of Pennsylvania showing the location of Tytoona Cave and Arch Spring (). 188 Northeastern Naturalist Vol. 20, No. 1 Figure 3. Entrance to Tytoona Cave. Tytoona Cave Nature Preserve is located in Sinking Valley, Blair County, PA. On the right side of the photo, the level of water of the stream at the time of the study can be seen. Fish were found throughout the cave along this stream. 2013 L. Espinasa, A. Mendyk, E. Schaffer, and A. Cahill 189 entrance to Tytoona Cave. Water from the spring flows into the surface stream of Sinking Run, which drains into the Little Juniata River, continues to the Juniata, and ultimately joins with the Susquehanna River. Tytoona Cave has a surveyed passage length of 1140 m, and is the 19th longest cave in Pennsylvania. When the surveyed passage in Arch Spring is added, the total known length of the system is approximately 1780 m. The estimate of a 50-m connection separating the explored sections of Tytoona and Arch Spring caves has been made via dye tracing. This sections remains to be dived, surveyed, and mapped. A detailed description of the cave and map of Tytoona/Arch Spring caves can be found in White and White (2012) and on the Tytoona Cave Nature Preserve Management Plan web page. Many of the cave’s passages are completely underwater with no air space (sumps), so humans can only explore them with scuba diving equipment. The current study was restricted to the area prior to the first sump between the entrance of the cave and the first 120 m, where there is a log jam . At the time of the study (3/31/12), the stream averaged around 3 m of width and 0.5 m in depth flowing quickly over gravel floors (Fig. 3), with a few calm pools at the sides. Sculpin were primarily observed in the main current, typically resting in eddies formed behind small rocks, but could also be found in the calm pools. Surface sculpins in Arch Spring and in Sinking Run were abundant and were typically found under rocks or mats of algae. Specimen collection Specimens were collected from Tytoona Cave, Arch Spring, the Sinking Run, and the Little Juniata. In agreement with the Preserve Management Chair and because this was a preliminary study conducted within a nature preserve, sculpins were released unharmed after initial capture and collection of data within the cave. Exceptions included surface specimens and a single cave specimen, which were preserved in 10% formalin. Sampling occurred on 30 and 31 March 2012. Specimens in the cave were located using headlamps and then collected with 40-cm-wide handheld bait nets. Specimens from the surface were collected using seines or handheld bait nets by disturbing rocks and algae. Standard length, head width, eye length, and pectoral fin length were measured with dial calipers to the nearest 0.1 mm. Mass was measured in the field with a portable balance to the nearest 0.01 g. Digital photographs of the body and the head, with the mandibular pores in the lateral plane on focus, were also taken while in the field. We measured mandibular pore #3 to the nearest 0.01 mm and assessed, from the digital photographs, if mandibular pore VI was a single pore (Fig. 1B), as in surface sculpin, or if there were two pores (Fig. 1A) as in some of the Pennsylvania Grotto Sculpins. Figure 4 (opposite page, bottom). Arch Spring is the resurgence of the Tytoona Cave system. Surface sculpin are abundant at this location. Excluding the conditions associated with the cave environment, such as continuous darkness, no major physical barrier exists between the resurgence and Tytoona Cave that a sculpin cannot overcome by swimming upstream. Humans using scuba have explored most of the galleries between the resurgence at Arch Spring and Tytoona Cave, with the exception of an estimated 50-m underwater connection (White and White 2012). 190 Northeastern Naturalist Vol. 20, No. 1 Statistical differences in the size of the eye, head, pectoral fin and mandibular pore #3 were assessed using linear regressions and t-tests. Dissection of the abdomen in the single lone cave specimen collected was done using a scalpel, dissection needles, and a dissection microscope to confirm if it was a gravid female. The distal edge of the right pectoral fin was cut from the captured cave specimens before being released for population-size estimation and genetic analyses. In agreement with the Preserve Management Committee, only one cave specimen could be extracted from the cave. As a result, it was difficult to precisely assess pigmentation differences between surface and cave specimens based on photos taken under unequal illumination conditions of flash in the cave and outdoor sunlight on the surface. Due to this factor, pigmentation results should be interpreted with caution. Population size Specimens were captured and marked on the first day by cutting the distal edge of the right pectoral fin. Specimens were released at the same place they were collected. The number of specimens with the distinctive blunted edge on the right pectoral fin recaptured on the second day was recorded and the following formula was used: Total = (original number tagged x total recaptured) ÷ number tagged on recapture. Sculpin are slow swimmers that lay motionless in the bottom of the stream for extended periods of time. Therefore, we assume that the 24 hrs allowed to pass between the two captures was sufficient for the marked individuals to redistribute themselves among the unmarked individuals only in close proximity. We also assume that this is a random mixture for only a very local population estimate and certainly not for the total population. Genetic analyses Tissue samples were deposited in 100% ethanol. Total DNA was extracted from fin clippings using the Qiagen DNEasy® Tissue Kit. Polymerase chain reaction was used to amplify and sequence the 16S rRNA mitochondrial marker using the 16Sar and 16Sb primer pair following protocols in Edgecombe et al. (2002). Amplification was carried out in a 50-μl volume reaction, with 1.25 units of AmpliTaq® DNA Polymerase (Perkin Elmer, Foster City, CA), 200 μM of dNTPs, and 1 μM of each primer. PCR products were purified with the Qiagen QIAquick® Gel Extraction Kit and directly sequenced using an automated ABI Prism® 3700 DNA analyzer as in Espinasa et al. (2007). Electropherograms obtained were read using the DNA sequence-editing software SequencherTM 3.0. Sequences were then compared between the cave fish and the surface fish and against published sequences found in the Genbank. Alignment was done with ClustalW. Results Morphology The Pennsylvanian Grotto Sculpins from Eiswert #1 Cave have a suite of modifications that readily identify them as cave-adapted: smaller eyes, elongated 2013 L. Espinasa, A. Mendyk, E. Schaffer, and A. Cahill 191 pectoral fins, broader heads and mouths, and more numerous and enlarged cephalic lateralis pores compared to surface sculpin. The Tytoona Cave population lacks most of this suite of characters. Eyes are as large as their surface counterparts (0.5 < P < 0.8), the width of their head is equivalent (0.2 < P < 0.5), and they lack the extra mandibular pore VI found in some Grotto Sculpins; therefore, the number of cephalic lateralis pores is the same as in the surface fish. The cave specimens even have shorter instead of longer pectoral fins when compared with their surface counterparts (P < 0.001), although this result should be considered with caution because it was noticed that the cave specimens had some type of fin infections that shredded their fin tips. Nonetheless, we were able to document a distinct difference in the Tytoona population. Cephalic lateralis pore size (Fig. 1) is a clear discriminating feature between Tytoona Cave fish and surface fish (Fig. 5). The cave population has distinctly larger pore size than similar-sized surface fish (P < 0.001). Mandibular pore III of cave sculpin was approximately 20% larger than those from surface specimens. Both Tytoona cave and surface sculpin show considerable variability in pigmentation and color patterns (Fig. 6). Cave sculpin are not albino and have well-developed pigmentation. Nonetheless, the previously described Pennsylvanian Grotto Sculpin from Eiswert #1 Cave, which is considered cave-adapted, are also not albino and have variable pigmentation levels as well. In contrast to the sculpins in Eiswerth #1, where the extreme lowest pigmented specimens are clearly depigmented when compared with surface specimens, sculpin in Tytoona Cave do not appear to have that level of depigmentation. A few cave specimens Figure 5. Mandibular pores III of the Tytoona Cave fish are about 20% larger than those from surface samples of equivalent standard length (SL). Cephalic lateralis pore size is a clear discriminating feature between cave fish and surface fish. The cave population has distinctly larger pore size than similar-sized surface fish (P < 0.001). 192 Northeastern Naturalist Vol. 20, No. 1 may be beyond the lowest levels of pigmentation of sculpin found in adjacent surface habitats. On the date of collection (30–31 March), it was evident that many specimens, both from the surface and the cave, had distended abdomens (Fig. 6B). Dissections made of several surface specimens and of the single preserved cave specimen showed they were females carrying eggs. Since a single cave female was available, sample size is inadequate to statistically assess if the number and size of eggs is different from the surface, although in appearance they were within the normal range of the surface specimens dissected. The existence in the cave of ovigerous females and of individuals spanning a broad range of sizes (2.2–7.4 cm) supports that this may be a population breeding wi thin the cave. Weight The weight per unit of length can reveal much about the condition of a fish population inhabiting a particular environment. Accidental, non-cave-adapted organisms found in caves are often lean as a result of weight loss due to starvation (Hervant 2012). The Tytoona Cave population shows no evidence of being in a different condition than that of their surface counterparts. Their weight per unit of length is nearly identical to the surface specimens (Fi g. 7). Figure 6. Variability of pigmentation and color patterns in the Tytoona Cave sculpin. While most cave-fish pigmentation levels are within the range of surface fish (A and C), some may be less pigmented (B). Specimen B is also an ovigerous female, as seen by the distended abdomen. 2013 L. Espinasa, A. Mendyk, E. Schaffer, and A. Cahill 193 Population size Fifteen specimens were marked on the first day, and only two of these were recaptured on the second day, when eighteen specimens were collected. This resulted in an estimate of a local population size of 135 indiv iduals. The estimated 135 local individuals came from about only 120 m of gallery because we assume that the 24 hrs allowed to pass between the two captures was sufficient for the marked individuals to redistribute themselves among the unmarked individuals in close proximity to give a random mixture for only a very local population estimate and certainly not for the total population. The river passage to the spring has a minimum of 1220 m, and therefore, it is reasonable to expect that the fish population is composed of hundreds and maybe even thousands of individuals. DNA sequences Molecular data have been obtained for 15 individuals: 14 samples from Tytoona Cave and one from a surface specimen from the Little Juniata River. The 16S rRNA fragments with primers excluded were 575 bp long. All cave and surface specimens had identical sequences, which when compared with the available Genbank sequences, were identical to “haplotype 2” of a Cottus bairdi collected from Blockhouse Creek in central Pennsylvania (GenBank# GQ280792.1) and four bp (0.7%) different from another C. bairdi of undescribed provenance (Gen- Bank# AY539018.2). Discussion Reports from local cavers indicate that Tytoona Cave in Central Pennsylvania is inhabited by a population of sculpins. The relevant question to be answered is: are they simply surface fish that have somehow ended up inside the cave, or is Figure 7. Tytoona Cave fish has the same weight as those from surface samples of equivalent standard length (SL). No evidence for starvation is present. It appears that the cave population is efficient in obtaining energy from the food resources available in the cave. This finding implies that the Tytoona population may be sufficiently adapted to exploit the resources of the cave niche. 194 Northeastern Naturalist Vol. 20, No. 1 this a unique, cave-adapted population? If proven cave adapted, it would be the second northernmost cave-adapted fish in the world. In this study, we show that Tytoona Cave is inhabited by a population of sculpin whose mitochondrial DNA place them within the Cottus bairdi group. The characters normally used in recognizing troglomorphic fish, reduction or loss of eyes and pigmentation, are not pronounced in the Tytoona Cave fish. This finding by itself, however, is not significant. The two previously described populations of cave-adapted sculpin, the Pennsylvanian Grotto Sculpin (Espinasa and Jeffery 2003) and the Missouri Grotto Sculpin (Burr et al. 2001), both have eyes and pigment, but are still considered cave-adapted. The reason is that, for example, the sculpins from the Nippenose Valley caves have a suite of morphological traits that readily identify them as cave-adapted: smaller eyes, elongated pectoral fins, more numerous and enlarged cephalic lateralis pores, a broader head, reduced average pigmentation, degenerated retinas, increased subdermal fat reserves, and a size reduction of the tectum opticum in the brain (Espinasa and Jeffery 2003). Not all of the aforementioned characters could be determined in the Tytoona Cave population because they require sacrificing multiple specimens. In agreement with the Tytoona Cave Nature Preserve Management Committee, gathering of data was immediately followed by release of all captured specimens, with the exception of a single individual. Therefore morphology of retina, subdermal fat reserves, and brain features were not examined. Overall results should be considered preliminary due to the intrinsic difficulties of measuring in the field under the trying conditions of total darkness and high humidity insid e a river cave. Data shows that the Tytoona Cave population lacks most of the aforementioned suite of modifications. They have eyes as large as their surface counterparts, the length of pectoral fins and head is equivalent, they have the same number of cephalic lateralis pores and the degree of pigmentation is similar. While the above data would support that they are simply surface fish that entered the cave, there is also evidence that this is truly a cave-adapted population. Surface organisms that accidentally end up in caves are typically starving and cannot reproduce inside the cave environment. Cave-adapted organisms, on the contrary, can thrive and reproduce. In the Tytoona Cave population, the absence of evidence for starvation and the presence of ovigerous females supports that this is a population adapted to survive and reproduce in the cave. But of highest relevance, they also appear to be morphologically distinct, having enhanced mechanosensory systems. The cephalic lateralis pores, which are part of the lateral line system, are significantly larger than similar-sized sculpin from adjacent surface habitats. Mandibular pores of the cave sculpin are about 20% larger than those of surface sculpin. These enlarged pores may enhance the mechanosensory detection of small water vibrations, such as those from prey that need to be detected in the darkness of the cave. It would be interesting to see if the population near the entrance of Arch Spring, where cave and surface fish may be in contact, show a gradient of mandibular pore size, but to collect in this area requires scuba diving techniques, which was beyond the means of this study . 2013 L. Espinasa, A. Mendyk, E. Schaffer, and A. Cahill 195 While the Tytoona Cave sculpin can be considered troglophiles because they are able to thrive and reproduce in the cave environment, they certainly have fewer troglomorphic characters than those of previously reported cave-adapted sculpins (Burr et al. 2001, Espinasa and Jeffery 2003). By comparison, the Tytoona Cave population has only some emergent cave adaptations, reflecting either a more recent divergence from surface C. bairdi, or that gene flow is still present. Gene flow into a population can counteract the effects of selection on gene frequency, imposing a limit on local adaptation (Lenormand 2002). If there is gene flow between the surface sculpin and the Tytoona population, it could be preventing the differentiation of characters except for those that are most selectively essential for survival inside the cave. As for a recent divergence, in high latitudes, the extent of polar ice sheet migration during the Pleistocene era has restricted colonization of caves until at least 12 ka ago, when the region was no longer covered by ice sheets. The lack of belowground species in northern latitudes, despite suitable habitats, might be attributed to extinctions during Pleistocene glaciations and the inability of rangerestricted taxa to re-colonize these regions (Schuldt and Assmann 2011). As a result, available evolutionary time has been limited for northern cave fish. It may be that the available time, restriction of gene flow, and/or strength of other evolutionary forces have been sufficient enough to enhance mechanosensory systems, but have yet to regress eyes and pigmentation in the Tytoona Cave population. Finally, our study indicates a healthy population size of hundreds or potentially even thousands of individuals. For conservation purposes, the population does not appear to be at imminent risk, but results of this population size pilot study should be interpreted with caution, as its design was not to obtain the actual total population size, but only to provide a baseline on which the Preserve Management Committee could better assess the impact of biological studies on this population. Acknowledgments We thank Garrett Czmor, Tytoona Cave Nature Preserve Management chair, for providing the permit to study the cave population at Tytoona Cave. 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