Species Profile: Alabama Cavefish (Speoplatyrhinus poulsoni)

Alabama Cavefish adult (Photo by Dante Fenolio)

Alabama Cavefish
Speoplatyrhinus poulsoni

Conservation status: IUCN Red List - Critically Endangered C2b; NatureServe - G1 (Alabama: S1). Listed as Endangered under the U.S. Endangered Species Act. Listed as Endangered in Alabama.

Description: Speoplatyrhinus poulsoni is an eyeless and depigmented amblyopsid cavefish that is pinkish-white in color with some structures, like fins, fin rays and the venter, that are quite translucent. Morphologically, it is the most cave-adapted fish in the family Amblyopsidae. Adults are typically 30-58 mm (1.2-2.3 inches) standard length (SL). Both adults and juveniles have an extremely elongate and flattened snout with a terminal mouth that is duck-like in appearance. Unlike other amblyopsid cavefishes, S. poulsoni lacks branched fin rays, and the fin membranes are incised giving a spiked appearance. Pelvic fins are lacking. Fin rays counts are as follows: 9 (9-10) dorsal, 8 (8-9) anal, 9 (9-11) pectoral, and 22 (21-22) caudal. The lateral-line system is hypertrophied and their is an elaborated system of superficial neuromasts arranged in distinct ridges on the head and along the body. Caudal sensory papillae are also found on the caudal fin. Scales are small, imbedded, and cycloid. The urogenital pore and anus are jugular in position. Recent molecular work indicates that S. poulsoni is most closely related to the Southern Cavefish (Typhlichthys subterraneus).

Lateral view of an Alabama Cavefish.

Distribution: Speoplatyrhinus poulsoni is known from just a single cave system, Key Cave, in Lauderdale Co., Alabama, within the Tennessee River watershed. Despite numerous surveys for cavefishes in other caves in close proximity to Key Cave along the Tennessee River and elsewhere, no additional populations of S. poulsoni have been found in northwestern Alabama.

Habitat: Key Cave is a maze-like cave system developed in the Mississippian-aged Tuscumbia Limestone. The aquatic habitat in Key Cave consists of a series of pools with little flow that occur in a zone of seasonal oscillation of the local water table. Several of these pools are quite deep reaching depths of up to 5 m depending on seasonal water levels. Significant bat roosts occur near at least two pools where guano occasionally slides or falls into the water.

Close-up dorsal view of head.

Natural History: Little is known regarding many aspects of the life history and ecology of S. poulsoni because of its rarity. Some authors have speculated that females may incubate eggs and protect young fry within the branchial chamber, based on the jugular position of the vent. This behavior has been observed in some populations of the related Northern Cavefish (Amblyopsis spelaea) but has yet to be demonstrated for any other amblyopsid, including S. poulsoni. Individuals as small as 12-15 mm SL have been observed in February and November, suggesting that S. poulsoni may breed in the summer months. In addition, the female holotype contained developing ova and was collected in late May, also supporting a summer spawning season. The diet of S. poulsoni has not been studied but likely includes copepods, isopods, amphipods, and perhaps small crayfish. An undescribed species of cave shrimp was recently found in Key Cave and likely is prey for S. poulsoni. Predators are unknown and its thought that S. poulsoni is one of the top consumers in the Key Cave ecosystem. Sympatry of cavefish species is rare; however, S. poulsoni cooccurs with T. subterraneus within Key Cave. Typhlichthys subterraneus is common in caves throughout central Kentucky, central Tennessee, northern Alabama, and extreme northwestern Georgia. The absence of S. poulsoni but presence of T. subterraneus from nearby cave systems suggest that competitive interactions might influence the distribution of S. poulsoni. However, this hypothesis has not been examined.

Dorsal view of an Alabama Cavefish.

Conservation: Speoplatyrhinus poulsoni is one of the rarest vertebrates in North America. The population in Key Cave is small and no more than 10 individuals have ever been observed during a single survey.The U.S. Fish and Wildlife Service listed S. poulsoni as threatened in 1977 based on its restricted distribution, low abundance and potential threats to this species, including disturbance of a maternity colony of endangered Gray Bats (Myotis grisescens) whose guano is an important source of nutrients and energy for the aquatic ecosystem in Key Cave and groundwater pollution from pesticides as well as a proposed industrial park for the city of Florence. The threat status was later reclassified as endangered in 1988, as a sewage sludge disposal operation was found to occur within the recharge area of Key Cave. In addition, herbicide and pesticide runoff from cotton fields was found to have direct access into Key Cave via surface seeps. Loss of aquatic habitat from lowering of local groundwater levels by increased pumping also has been cited as a concern. The U.S. Fish and Wildlife Service purchased several hundred acres of land within the recharge zone of the cave and established the Key Cave National Wildlife Refuge. The agricultural land within the refuge is still maintained but cotton was replaced with corn and soybeans. In addition, all chemical use was restricted. This agricultural land is slowly being converted to upland forest and native grasslands. The most recent surveys for S. poulsoni indicate that the population is stable and recruitment is still occurring.

Fun Fact: Speoplatyrhinus poulsoni is named in honor of Dr. Tom Poulson, a prominent cave biologist who has studied amblyopsid cavefishes and other cave life since the late 1950s.

Select References

Boschung HT, Mayden RL. 2004. Fishes of Alabama. Smithsonian Institution Press, Washington.

Cooper JC, Kuehne RA. 1974. Speoplatyrhinus poulsoni, a new genus and species of subterranean fish from Alabama. Copeia 1974: 486-493.

Kuhajda BR. 2004. The impact of the proposed Eddie Frost Commerce Park on Speoplatyrhinus poulsoni, the Alabama cavefish, a federally endangered species restricted to Key Cave, Lauderdale County, Alabama. Endangered Species Update 21: 57.

Kuhajda BR, Mayden RL. 2001. Status of the federally endangered Alabama cavefish, Speoplatyrhinus poulsoni (Amblyopsidae), in Key Cave and surrounding caves, Alabama. Environmental Biology of Fishes 62: 215-222.

Niemiller ML, Near TJ, Fitzpatrick BM. 2012. Delimiting species using multilocus data: diagnosing cryptic diversity in the southern cavefish Typhlichthys subterraneus (Teleostei: Amblyopsidae). Evolution 66: 846-866.

Niemiller ML, Poulson TL. 2010. Subterranean fishes of North America: Amblyopsidae. Pp. 169-280 in: Trajano E, Bichuette ME, and Kapoor BG (eds). The biology of subterranean fishes. Science Publishers, Enfield, New Hamphire.

Poulson TL. 2009. New studies of Speoplatyrhinus poulsoni (Pisces: Amblyopsidae). Proceedings of the 15th International Congress of Speleology 3: 1337-1342.

Proudlove GS. 2006. Subterranean fishes of the world. International Society for Subterranean Biology, Moulis, France.

Romero A. 1998. Threatened fishes of the world: Speoplatyrhinus poulsoni Cooper & Kuehne, 1974 (Amblyopsidae). Environmental Biology of Fishes 62: 293-294.

U.S. Fish and Wildlife Service. 1977. Final threatened and status and critical habitat for five species of southeastern fishes. Federal Register 42: 45526-45530.

U.S. Fish and Wildlife Service. 1982. Recovery plan for the Alabama cavefish, Speoplatyrhinus poulsoni Cooper and Kuehne 1974. Prepared by Cooper JE, North Carolina State Museum of Natural History. 72pp.

U.S. Fish and Wildlife Service. 1988. Endangered and threatened wildlife and plants: reclassification of the Alabama cavefish from threatened to endangered. Federal Register 53: 37968-37969.

U.S. Fish and Wildlife Service. 1990. Alabama cavefish, Speoplatyrhinus poulsoni Cooper and Kuehne 1974 (Second Revision) recovery plan. Prepared by Cooper JE, North Carolina State Museum of Natural History. Revised by Stewart JH, U.S. Fish and Wildlife Service, Atlanta, Georgia. 17pp.

Loss of function in the eye gene rhodopsin of amblyopsid cavefishes

Figure 1. Time-calibrated phylogeny of Percopsiformes including cave and surface species of amblyopsid cavefishes inferred from a multilocus dataset. Taxa in gray are cave lineages. Loss of function (red) and nonsynonymous (white) mutations in the photoreceptor gene rhodopsin are indicated on the branches. Cavefish images courtesy of Dante Fenolio.

Troglobites (obligate cave-dwelling organisms) are population examples of regressive evolution, the repeated degeneration or loss of derived traits. For example, many independent lineages have lost their eyes and are depigmented, such as millipedes, crustaceans, fishes, and salamanders. These phenotypic changes are thought to have evolved in response to the challenging environmental conditions found in subterranean habitats, particularly the absence of light.

Despite the long recognition of regressive evolution in subterranean organisms, there has been surprisingly little evidence for regressive evolution at the molecular in genes associated with regressive characters (e.g., genes involved in the development and function of the eye). The best examples include studies of eye genes in subterranean diving beetles [1], marsupial moles [2], naked mole rats [3], Astanyax cavefish [4], and cave-roosting bats [5]. Several studies of perhaps the most well-studied eye genes, the retinal photoreceptor gene rhodopsin, in subterranean organisms have yet to uncover compelling evidence for loss of gene functionality at the molecular level. For example, Kim et al. [3] discovered at least 19 genes associated with visual perception that were either lost or showed evidence for loss of function in the naked mole rat but rhodopsin was not one of these loci.

What might account for apparent functionality of rhodopsin in the subterranean organisms studied previously? One hypothesis is weak selection for the retention of light-sensing abilities [3, 6]. Another possible explanation is that rhodopsin has other pleiotropic functions, such as in circadian rhythms [7-9]. Support for this latter hypothesis was demonstrated in a recent study by Shen et al. [10] who discovered that invertebrate rhodopsin was important in initiating thermosensory-signaling cascades in Drosophila fruit flies.

An alternative hypothesis to explain presumed functionality in rhodopsin is that the gene no longer has a function in aphotic environments but insufficient time has passed for the accumulation of loss of function (LOF) mutations to accumulate and render the gene nonfunctional. This hypothesis assumes that selection is not the mechanism behind loss of functionality [1]. If neutral processes are responsible for loss of functionality, then integrity of a gene may persist for a considerable period of time due to chance alone [11]. Consequently, inferred maintenance of a visual gene may be the result of recent subterranean colonization rather than retained or pleiotropic functionality.

My colleagues (Drs. Ben Fitzpatrick, Premal Shah, Lars Schmitz and Tom Near)  and I investigated this latter hypothesis by testing for loss of selective constraint in rhodopsin of amblyopsid cavefishes in a recently accepted paper in the journal Evolution [12]. We first generated a fossil-calibrated, multilocus molecular phylogeny based on nine genes to provide a temporal and phylogenetic context for elucidating the evolutionary history and potential loss of rhodopsin functionality. Our sampling included every recognized species of amblyopsid cavefish (family Amblyopsidae) as well as related outgroup taxa within the order Percopsiformes (families Aphredoderidae and Percopsidae). The resulting phylogeny (Figure 1) differed considerably from previously phylogenetic hypotheses [13-15], with the most notable difference being the phylogenetic placement of the surface/facultative cave-dwelling genus Forbesichthys. Forbesichthys was nested within a clade of all obligate cave-dwelling lineages and diverged some 5.7 Mya. The most recent common ancestor (MRCA) of all amblyopsids (cave and surface lineages) dates to 12.2 Mya in the Miocene while the MRCA of all cave lineages dates to 10.3 Mya. However, much of the diversification within genera occurred in the Pleistocene.

The intriguing phylogenetic placement of Forbesicthys suggests that a surface-dwelling life history and eye functionality may have reevolved from a cave-dwelling ancestor with degenerate eyes. In fact, ancestral character reconstructions of eye functionality in amblyopsid cavefishes strongly support reevolution in Forbesichthys from a subterranean ancestor. Cave-dwelling organisms are often viewed as "evolutionary dead-ends" that are incapable of recolonizing or adapting to surface habitats. However, the reevolution of a surface form is not novel, as this hypothesis has been proposed for eyed Gammarus amphipods living in karst windows [16] as well as in cave scorpions [17], cavefishes [18] and cave salamanders [19].

Figure 2. Rhodopsin gene tree of percopsiform fishes with nonsynonymous mutations (white square), deletions (red square), insertions (green square), and premature stop codons (red octagon) mapped onto branches. Cave lineages are in gray. The size of indels (in base pairs) is indicated within the red or green square. The inset shows two-dimensional models of rhodopsin with cumulative nonsynonymous mutations, deletions, insertions, and premature stop codons indicated for surface and cave lineages.

However, patterns of molecular evolution in rhodopsin suggest a different evolutionary history (Figure 2). We found no evidence of loss of selective constraint or functionality in rhodopsin of surface lineages, which was to be expected given that surface-dwelling species actively respond to photic stimuli. However, three cave lineages (Troglichthys rosae, Amblyopsis spelaea and Typhlichthys cf. subterraneus TN) exhibited strong evidence for loss of rhodopsin functionality at the molecular level including ten novel LOF mutations (six deletions, one insertion, and three mutations resulting in premature stop codons). These LOF mutations were highlighted by an 111 amino acid deletion in the federally listed Troglichthys rosae. In addition, rhodopsin in cave lineages (even those without LOF mutations) showed increased rates of nonsynonymous (amino acid changing) mutations compared to surface lineages. These mutations were more likely to have a significant deleterious effect on the structure and subsequently function of rhodopsin in cave lineages, as inferred from several physicochemical protein properties.

As a final test to determine if rhodopsin in cave lineages was evolving at a different evolutionary rate (i.e., under neutrality versus balancing or positive selection) compared to surface lineages, we compared a series of branch-based models of selection using the multilocus phylogeny. Interestingly, the best model corresponded to  independent evolution of relaxation of selection and loss of functionality in cave lineages rather than reevolution of rhodopsin functionality in Forbesichthys that was suggested based on ancestral character reconstructions. In fact, not a single nonsynonymous mutation was observed in the 4.6 My interval from the MRCA of all cave amblyopsids and the MRCA of Forbesichthys and Amblyopsis (node b-e in Figure 1), as would be expected if there was a single subterranean colonization event and subsequent reevolution of eyes in Forbesichthys. Other lines of evidence also support independent evolution of loss of eye (and rhodopsin) functionality over a reevolution scenario, including patterns of degeneration of individual eye structures based on histological data, biogeographical and geological evidence, and phylogenetic studies of other cave organisms.

In summary, our study provides compelling evidence for repeated loss of selective constraint in rhodopsin in cave amblyopsids. Although we cannot rule out selection as the primary mechanism behind eye degeneration (i.e., selection might act upstream of rhodopsin or at other important eye developmental loci), our results are consistent with the neutral accumulation of mutations responsible of loss of functionality in rhodopsin. Rather than some unknown pleiotropic function, we hypothesize that presumed functionality of rhodopsin in other cave organisms is due to recent subterranean colonization and the random nature of mutation accumulation.

References

1. Leys R, Cooper SJB, Strecker U, Wilkens H. 2005. Regressive evolution of an eye pigment gene in independently evolved eyeless subterranean diving beetles. Biol. Lett. 1: 496-499.

2. Springer MS, Burk A, Kavanagh JR, Wadell VG, Stanthope MJ. 1997. The interphotoreceptor retoid binding protein gene in therian mammals: implications for higher level relationships and evidence for loss of function in the marsupial mole. Proc. Natl. Acad. Sci. USA 94: 13754-13759.

3. Kim EB et al. 2011. Genome sequencing reveals insights into physiology and longevity of the naked mole rat. Nature 479: 223-227.

4. Yokoyama S, Meany A, Wilkens H, Yokoyama R. 1995. Initial mutational steps toward loss of opsin gene function in cavefish. Mol. Biol. Evol. 12: 527-532.

5. Zhao H, Rossiter SJ, Teeling E, Li C, Cotton JA, Zhang S. 2009. The evolution of color vision in nocturnal mammals. Proc. Natl. Acad. Sci. USA 106: 8980-8985.

6. Culver DC, Pipan T. 2009. Biology of Caves and Other Subterranean Habitats. Oxford University Press.

7. Crandall KA, Hillis DM. 1997. Rhodopsin in the dark. Nature 387: 667-668.

8. Janssen JWH et al. 2000. A fully functional rod visual pigment in a blind mammal. A case for adaptive functional reorganization? J. Biol. Chem. 275: 38674-38679.

9. Li Z, He S. 2009. Relaxed purifying selection of rhodopsin gene within a Chinese endemic cavefish genus Sinocyclocheilus (Pisces: Cypriniformes). Hydrobiologia 624: 139-149.

10. Shen WL, Kwon Y, Adegbola AA, Luo J, Chess A, Montell C. 2011. Function of rhodopsin in temperature discrimination in Drosophila. Science 331: 1333-1336.

11. Marshall CR, Raff EC, Raff RA. 1994. Dollo's law and the death and resurrection of genes. Proc. Natl. Acad. Sci. USA 91: 12283-12287.

12. Niemiller ML, Fitzpatrick BM, Shah P, Schmitz L, Near TJ. In press. Evidence for repeated loss of selective constraint in rhodopsin of amblyopsid cavefishes (Teleostei: Amblyopsidae). Evolution.

13. Woods LP, Inger RF. 1957. The cave, spring, and swamp fishes of the family Amblyopsidae of central and eastern United States. Am. Midl. Nat. 58: 232-256.

14. Swofford DL. 1982. Genetic variability, population differentiation, and biochemical relationships in the family Amblyopsidae. Master's thesis. Eastern Kentucky University, Richmond, KY.

15. Niemiller ML, Poulson TL. 2010. Subterranean fishes of North America: Amblyopsidae. Pp. 169-280 in Trajano E, Bichuette ME, Kappor BG (eds.). The Biology of Subterranean Fishes. Science Publishers, Enfield, NH.

16. Culver DC, Kane TC, Fong DW. 1995. Adaptation and Natural Selection in Caves: the Evolution of Gammarus minus. Harvard University Press, Cambridge, UK.

17. Prendini L, Francke OF, Vignoli V. 2010. Troglomorphism, trichobothriotaxy and typhlochactid phylogeny (Scorpiones, Chactoidea): more evidence that troglobitism is not an evolutionary dead-end. Cladistics 26: 117-142.

18. Dillman CB, Bergstrom DE, Noltie DB, Holtsford TP, Mayden RL. 2011. Regressive progression, progressive regression or neither? Phylogeny and evolution of the Percopsiformes (Teleostei, Paracanthopterygii). Zool. Scr. 40: 45-60.

19. Trajano E, Cobolli M. 2012. Evolution of lineages. Pp. 295-304 in White WB, Culver DC (eds.). Encyclopedia of Caves, 2nd ed. Academic Press, Oxford.