The habitat of cave-adapted organisms is characterized by complete darkness and in some instances, an apparent lack of environmental distinction between day and night. It is unclear if cave-adapted organisms retain circadian rhythms that can be light-entrained. Stygobromus allegheniensis (Allegheny Cave Amphipod) is an eyeless troglobitic crustacean found in caves located in the Northeastern region of the United States. Two cave populations were examined for evidence of light-entrained circadian rhythms. The first population inhabits a small tectonic cave (Ice Caves, Sam’s Point Preserve, NY) and the second (Clarksville Cave, Clarksville, NY) inhabits a long cave system in limestone rock. Experiments conducted in both the field and the laboratory suggest that the capacity to exhibit motor rhythms has been conserved in at least some individuals of both populations. Nonetheless, their motor activity rhythms have high variability of period length between individuals and do not appear to be light-entrainable. It is thus proposed that in this species, light-entrainable circadian rhythms controlling motor activity have undergone incipient regressive evolution.

Stygobromus allegheniensis , Shawangunk, nyctophilia, Ice Caves, Sam’s Point Preserve, Clarksville Cave, troglobite, troglobiont, light-entrainment

In the evolutionary history of organisms, the loss or reduction of ancestral characters is a common event that occurs when a character is no longer needed for survival. Examples of this phenomenon in nature include the regression of pelvic and posterior appendages in whales (Bejder and Hall 2002), teeth in birds (Chen et al. 2000), and eyes and dermal pigmentation in cavefish (Jeffery 2001; McCauley et al. 2004). Cave organisms are an excellent model in which to study regressive evolution. Species from many different lineages have undergone parallel evolution and have developed similar traits such as blindness, depigmentation and the loss of circadian rhythms due to the constraints of living in continuous darkness.

Despite the scientific community’s interest in chronobiological research in species that do not experience day and night cycles, and the fact that cave-dwelling animals represent a powerful model for understanding the evolution of biological rhythms, few studies have been conducted in this field. Pasquali and Sbordoni (2014) and Beale and Whitman (2015) reviewed the literature and suggest that circadian activity tends to degenerate in cave obligate organisms, as indicated by comparisons of epigean and troglophilic species to troglobitic ones. However, the data have shown great variability. To contribute to a better understanding of this important aspect of regressive evolution, we have studied motor rhythms in the eyeless cave amphipod, Stygobromus allegheniensis Holsinger, 1967.

Stygobromus allegheniensis is a completely depigmented and eyeless amphipod, with the largest individuals reaching 2 cm long (Figure 1). Despite the complete regression of ocular structures, this species is able to detect light (Espinasa et al. 2015). Individuals from this species exhibit nyctophilia, a distinct preference for darkness over illuminated conditions. Furthermore, their non-ocular receptors appear to be tuned to specific wavelengths of light, as specimens do not respond to red light, but actively avoid green light (Espinasa et al. 2015).

Figure 1. 

Adult and juvenile specimens of Stygobromus allegheniensis from Ice Cave #1 at Sam’s Point Preserve. As is typical of cave-adapted organisms, this species is depigmented, has long appendages, and is fully eyeless. Nonetheless, it can detect light and actively avoids it.

This species is located within caves found in Maryland, Pennsylvania, and New York, covering a distance of approximately 596 km from North to South, making it one of the largest ranges of any troglobiont in this genus (Holsinger 1967). Despite its wide range, no significant morphological variation among populations has been found (Holsinger 1967), and DNA sequencing of Ice Caves and Clarksville Cave specimens supports that these distant populations belong to a single species (Cahill et al. 2015). The Ice Caves are found along the Shawangunk Ridge, at Sam’s Point within the Minnewaska State Park, New York, USA. Clarksville Cave is a popular horizontal stream cave with about 1.5 km of passage in Albany County, New York. Colonization of these caves by the amphipods occurred less than 12,000 years ago as both caves were covered by glacial ice sheets during the Pleistocene Epoch (Espinasa et al. 2015).

While the regression of morphological structures may occur over time in the absence of specific selective pressures, it is unclear if the physiological processes associated with the degenerated organs are maintained throughout these processes of regression. For example, it is unclear if light-entrained circadian rhythms still coordinate diverse, complex physiological processes including behavior when ocular structures are regressed in cave environments (Espinasa and Jeffery 2006). In S. allegheniensis, regression of the eyes has occurred, but remains able to detect and react to light. It is thus unclear if other responses to light, such as a light-entrainment of circadian rhythms, are still present. Therefore, the purpose of this study is to analyze if light entrainement of circadian motor rhythms has experienced regressive evolution in S. allegheniensis.

Experiments were conducted both in the field and in the laboratory using collected specimens of S. allegheniensis. Two cave populations were studied: a) Sam’s Point Ice Cave #1. Total length = 138 m. Minnewaska State Park Preserve, near Sams Point Rd., Cragsmoor, NY. October 24-25, 2015 for laboratory experiments and September 8–11, 2016 for field experiments. b) Clarksville Cave. Total length = about 1,500 m. Clarksville, Albany County, NY. June 23–25, 2016.

Laboratory experiments

To test whether periods of activity or rest observed in the field followed light-entrainable circadian rhythms, specimens were brought to the laboratory and kept in darkness until the experiments were performed 60 hours post collection. Specimens were placed in separate 10 cm wide petri dishes with water to a height of 3 cm. Water from collection site was used. Two 100W light bulbs positioned one meter above the tanks were used for illumination during light periods. Previous studies (Espinasa et al. 2015) performed under similar illumination conditions have shown that the temperature difference between the beginning of the experiment and the end of the experiment is not significantly different. Data analyses were done with the customary experimental set-ups for studying circadian rhythms, which include: 1) Subjecting specimens to continuous conditions (such as constant darkness) to test if motor activity follows a 24 hour rhythmicity. 2) Subjecting specimens to conditions of light-dark-light-dark-dark (LDLDD 14:10:14:10:14) as organisms following a light-entrained circadian rhythm are expected to behave in the last second dark period in similar ways to those of the light period as the internal clock is anticipating a period of illumination (Espinasa and Jeffery 2006). 3) Transferring from a day/light, night/dark periodic environment to a day/dark, night/light environment to see if behavior adjusts to the new illumination pattern. Thus, three specimens from the Ice Caves were subjected to the following conditions: Five half-cycles (60 hrs) of darkness, followed by two cycles of light/dark during normal day/night schedules, followed by two cycles of dark/light during reverse day/night schedules, followed by two half-cycle of darkness (Figure 2). One cycle is twenty-four hrs. Three different specimens from the Ice Caves were also subjected to four cycles of continuous light. From Clarksville Cave, four specimens were subjected to six half-cycles of darkness, followed by two cycles of light/dark during normal day/night schedules, followed by one cycle of dark during the day. One cycle is twenty-four hours. Recording of movements and statistical analyses were performed as above.

Figure 2. 

Experimental protocol and representative motor rhythms of one individual. Ice Cave individuals were subjected in the laboratory to the following conditions: Five half-cycles of darkness, followed by two cycles of light/dark during normal day/night schedules, followed by two cycles of dark/light during reverse day/night schedules, followed by a half-cycle of darkness. Black boxes indicate dark conditions while white boxes represent illuminated conditions. Movements were evaluated for each 10-minute period.

To better understand the regressive evolution of circadian rhythms, we have studied the motor rhythms of eyeless cave amphipods who in their natural environment are submitted to continuous darkness, and thus reduced environmental distinction between the day and the night. Results from both the field and the laboratory suggest that S. allegheniensis has a degenerated expression of circadian rhythms. While specimens from the two cave populations have periods of intense movement followed by periods of apparent rest where specimens lay on their side with no body movements for extended periods, the periodicity of their motor rhythms are not synchronized to day and night cycles. This lack of synchronization would be expected in an environment that lacks distinction between day and night and in specimens with motor rhythms controlled by internal clocks that are not entrained by the environment. Furthermore, as it would be expected from an environment where selection for circadian rhythms is reduced, there is high variability in the length of the motor rhythms observed within a population. Some individuals appear to have motor-activity cycles that follow rhythms shorter than 24 hours while others follow rhythms that are longer (Figure 3). Most relevant, however, in seven out of thirteen (53.8%) specimens tested under continuous darkness for at least 36 hours there were no clear periods of rest of at least 6 hours followed by a period of activity of an equivalent length. This suggests that the motor periodicity may have been completely lost in some individuals (Figures 3A, D, 7A–D, F).

Despite the absence of functional eyes, experiments both in the field and in the laboratory show that light can generate motor activity. This is in agreement with Espinasa et al. (2015), which showed that individuals from this species actively try to avoid light. Nonetheless, the motor rhythms do not appear to be influenced by a light-entrained circadian rhythm. All specimens under conditions of light:dark:dark (LDD) failed to show activity in the second dark period that would normally be expected to mimic the activity observed in the light period (Figure 5). The anticipation and synchronization of a period of activity is lacking, which is a hallmark of organisms possessing a light-entrained circadian rhythm (Espinasa and Jeffery 2006). Likewise, specimens under continuous light conditions showed non-stop activity (Figure 6). Based on these results, light appears to generate continuous avoidance activity, but does not appear to be able to entrain circadian motor rhythms. It appears that in S. allegheniensis, light-entrainable circadian rhythms have undergone regressive evolution despite the retained ability to detect light.

Unfortunately, there are few circadian studies that have been performed on cave adapted animals for comparison. For a review of bibliography, see Beale and Whitmore (2015). One of these studies was on the cave amphipod, Niphargus puteanus Koch, 1836. Under continuous darkness (DD) regimes, the animals were active without clear pauses of rest, whereas light/dark (LD) conditions of various period lengths induced activity rhythms that usually disappeared after transfer back to constant conditions (Knight and Johns 2015). It appears that in N. puteanus there may not be motor rhythms when in complete darkness.

Beale and Whitman (2015) concluded that there is a vast array of circadian phenotypes in cave-adapted animals. Some retain partially functioning oscillators, some show highly variable rhythms between individuals within populations, and some show an absence of circadian rhythms all together. Our data suggest that S. allegheniensis may still have some partially functioning oscillators with highly variable rhythms between individuals, but which are not light-entrainable. It appears that while S. allegheniensis has undergone incipient regressive evolution of circadian rhythms, it has not reached the levels experienced by other cave adapted species where motor activity periodicity is completely lost. One hypothesis that could explain this level of regressive evolution is that S. allegheniensis may have a young and comparatively short cavernicole evolutionary history. The cavernicole habitat available for S. allegheniensis became available only when the glacial ice sheets retreated during the Pleistocene Epoch about 12,000 years ago (Espinasa et al. 2015). By comparison, some troglobites in more tropical areas unaffected by glaciation may have had millions of years for adaptation after colonization of the underground environment. Another possible explanation for S. allegheniensis is that this species is not strictly limited to the cave environment, but can also survive in the hyporheic zone under the gravel of surface creek beds. While still an underground environment, this habitat can be influenced by day and night periodicity.

We report a case of incipient regressive evolution of circadian rhythms in Stygobromus allegheniensis, an eyeless troglobitic crustacean found in caves located in the Northeastern region of the United States. While some individuals collected from two caves may display motor rhythms, they are no longer light-entrainable.

The research permit for the Ice Cave was issued by the science staff at the Palisades Interstate Park Commission, Jesse Jaycox and Ed McGowan. We would like to thank the staff at the Sam’s Point Area of Minnewaska State Park Preserve for their support, with special mention to Henry Alicandri. We would like to thank the Northeastern Cave Conservancy for granting us a permit to conduct research in Clarksville Cave and to the managers of Clarksville Cave Mike Chu, Chuck Porter and Thom Engel for permission to visit the cave. Bastien Dal Farra, Elsah Epstein, Abigail Descoteaux, Jae Wong, Michael Magin, and students of the Fall 2015 Field Biology (BIOL192) course helped in the field and/or analyzing the recorded videos. Partial support for the project came from the School of Science at Marist College.