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2013 Speleobiology Notes 5: 60–65. 60. Partial sequence of a gene involved in skin coloration (MC1R) from the Pennsylvanian Grotto Sculpin. Luis Espinasa1 ...

Espinasa et al.

Partial sequence of a gene involved in skin coloration (MC1R) from the Pennsylvanian Grotto Sculpin Luis Espinasa1, 2, Amy Cahill1, Sean McCaffery1 and Courtney Millar1 1

School of Science, Marist College, 3399 North Rd, Poughkeepsie, New York 12601, USA 2 [email protected] (corresponding author) Key Words: Cottus bairdi, Cottus cognatus, Cottidae, Scorpaeniformes. Actinopterygii, Eiswert #1 Cave, Nippenose Valley, Lycoming County, Pennsylvania, troglobite, MC1R, OCA2, brown phenotype, albinism, convergence.

In recent years, cave biology has received a significant impetus thanks to the relatively new field of Evolutionary Developmental Biology (Evo-Devo). Studies 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). These studies support that parallelism at the molecular level may be a common theme in the evolution of troglomorphic characters. Certain genes have been shown to be frequent targets of mutation in the repeated evolution of regressive phenotypes in cave-adapted species. While there are many genes that can tamper with the different steps in the metabolism of tyrosine to melanin and cause albinism, several populations of Blind Tetra (Astyanax) fish have independently undergone mutations in the same OCA2 gene. In one population there is a large deletion extending from within intron 23 through most of exon 24. In another Astyanax cavefish population there was another large deletion encompassing exon 21 that would also shorten the OCA2 protein. Both deletions are in regions predicted to be parts of membrane spanning domains and authors demonstrated that in some cave populations, the OCA2 channels were truly non-functional using a melan-P cell culture assay (Protas et al. 2006). A genetic defect at the first step of melanin synthesis is not restricted to Astyanax cavefish. Albinism is also caused by a defect in the first step of the melanin synthesis pathway in cave-adapted planthoppers from widely separated parts of the world in Hawaii and Croatia (Bilandžija et al. 2012). Furthermore, unpublished results show that this convergence in the specific first step of the metabolic pathway for melanin synthesis is also shared in cave nicoletiid insects and isopod crustaceans (H. Bilandžija and W. J. Jeffery, personal communication). This suggests convergent evolution of albinism in both cave-adapted arthropods and teleosts. 2013 Speleobiology Notes 5: 60–65  


Espinasa et al.

Another gene that is the frequent target of mutations in the repeated evolution of regressive phenotypes in cave-adapted fish is the melanocortin 1 receptor (MC1R). In mammals, the MC1R recessive variant gene results in red hair with fair skin color due to low concentrations of eumelanin throughout the body. In Astyanax cavefish, MC1R confers the Brown phenotype, independent from albinism. The brown phenotype affects eye color as well as the number and size of melanophores on the body (Sadoglu 1969), resulting in a decrease in total pigmentation. In some Astyanax cave populations, a 2 base-pair deletion in the extreme 5′ region of the open reading frame (positions 23 and 24) results in a non-functional transcript as it produces a frameshift that introduces a premature stop codon at nucleotide position 315. In other Astyanax populations there is a mutation at position 490 that causes an arginine to cysteine modification at amino acid position 164. Both alleles are likely to be amorphic, with the protein encoded by this locus being non-functional (Gross et al. 2009). The purpose of this study is to examine the MC1R gene in a different species of cave-adapted fish to determine if this gene is also a target of mutation in the repeated evolution of regressive phenotypes across cave-adapted species. The model used is the Pennsylvanian Grotto Sculpin. In 2003, Espinasa and Jeffery described the northernmost cave-adapted fish in the world. 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. Individuals of this cave population retain, although reduced, functional eyes and some pigmentation. Despite a spring with surface sculpin being only 445 m away from Eiswert # 1 Cave and an unlikely effective barrier of 10 m of altitude difference (Espinasa and Jeffery 2003), the cave fish are morphologically distinct from surface sculpin of the spring and nearby streams by their wider and more abundant mandibular pores, wider heads, longer pectoral fins, and reduced tectum opticum (area of the brain dedicated to vision). For the current study they are of particular interest because while they are not albino, they show a high degree of pigmentation variability, with some specimens being quite pale (Figure 1). Fish kept in the laboratory under illumination for up to three weeks retain, in comparison with surface fish, lower pigmentation levels, suggesting there is a genetic control of this character. The question being asked in this study is if there are variants of the MC1R gene in the sculpin cave population that may explain the depigmentation seen in these specimens. Since the description of the Pennsylvania Grotto Sculpins from Eiswert #1 Cave (41°9ʼ 23.2ʼʼN, 77° 12ʼ 21.1ʼʼW, 212 masl), these fish have been found at a second cave locality, Loose Tooth Cave (41°8ʼ 18.4ʼʼN, 77° 13ʼ 32.0ʼʼW, 223 masl), also within the Nippenose Valley. Sculpins were collected from both surface and caves with dip nets by James C. D. Lewis (Resident Pennsylvania Fishing License number R. 703557). Fin clips were deposited in vials with 100% ethanol. Tissue samples were obtained from: a) Cave samples: 19 individuals from Eiswert # 1 Cave (11 August 2002, 8 October 2002, 16 September 2007, 25 and January 2008) and two from Loose Tooth Cave (16 2013 Speleobiology Notes 5: 60–65  


Espinasa et al. September 2007); and b) Surface samples: Three individuals of C. cognatus Richardson, 1836 from Lochabar Spring, Antes Creek, Pennsylvania (41° 9ʼ 28.6ʼʼN, 77° 13ʼ 13.6ʼʼW, 200 masl; 8 October 2002 and 16 September 2007) and three of C. bairdi Girard, 1850 from Blockhouse Creek, Pennsylvania (41° 29ʼN, 77° 13ʼ W; 24 November 2002).

Figure 1. Dorsal view of Nippenose Valley troglomorphic (A–C) and surface Antes Creek (D) sculpins showing relative degrees of pigmentation: A) highly depigmented; B) slightly depigmented; C) pigmented; D) highly pigmented. Scale bar 2 cm. (Figure from Espinasa and Jeffery 2003). Genomic DNA was extracted using Qiagenʼs DNEasy Tissue Kit by digesting fin clips in lysis buffer. PCR amplification of a fragment of the MC1R gene was performed with the QIAGEN Multiplex PCR Kit® and primers GLISLVENI (forward: 5′GGGCCTGATCTCCCTGGTNGARAAYAT-3′) and IICNSLIDPL (reverse: 5′GGGGGTCGATCAGGGAGTTRCADATDAT-3′) as in Gross et al. (2009). Annealing temperature was of 48°C. PCR products were purified with QIAquick PCR Purification Kit® and directly sequenced using an automated ABI Prism® 3700 DNA analyzer. Chromatograms obtained from the automated sequencer were read and contigs were made using the sequence editing software SequencherTM 3.0. External primers were excluded from the analyses. Sequences were compared against GenBank sequences using Blast. 2013 Speleobiology Notes 5: 60–65  


Espinasa et al.

MC1R sequences were obtained from 21 cave specimens and six surface specimens. A fragment of 679 bp in length was amplified from the coding region (226 amino acids). The fragment amplified does not include the regions for the presumed first 61 amino acids nor the last 35 amino acids. All samples of Pennsylvania Grotto Sculpins, surface C. cognatus and surface C. bairdi had identical MC1R sequences (GenBank No. KC989742). Examination of chromatograms did not reveal any heterozygous sites. When this sequence was compared against sequences on GenBank, it showed a 99% maximum identity with the MC1R gene of Cottus gobio melanocortin 1 receptor gene (GenBank No. JX628000), The Pennsylvanian Grotto Sculpin, the northernmost cave-adapted fish in the world, is not albino, but shows a high degree of pigmentation variability, with some specimens appearing nearly depigmented (Figure 1). In this study we did not find support for melanocortin 1 receptor (MC1R), a gene that affects the concentrations of eumelanin throughout the body, as being the target of a mutation. While different mutations of the MC1R gene have arisen independently in geographically separate cave populations in Blind Tetras of the genus Astyanax (Gross et al. 2009), partial sequence of the coding region of this gene was identical in both surface and cave cottids. This may represent recent divergence of cave populations from surface populations. In high latitudes, the extent of polar ice sheet migration during the Pleistocene Epoch has restricted colonization of caves until at least 12 ka ago (Schuldt and Assmann 2011). As a result, evolutionary time has been limited for Pennsylvania Grotto Sculpins. 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 and regression of the eyes, but have yet to regress the MC1R in the Pennsylvanian Grotto Sculpin. Our results should be interpreted with caution, however. While we sequenced nearly 70% of the coding region of the MC1R gene and found no difference between cave and surface Cottus, there is still the possibility that mutations are present in the remaining 30% of the coding region or in the regulatory sequence of the gene. Sequencing these remaining areas was beyond the means of this preliminary study. Since there was no previous genetic information for this area in Cottus, degenerate primers that target the MC1R gene across species had to be used and these only amplified about 70% of the coding region of the gene. In addition, MC1R should be examined in other non-albino, troglomorphic cottid populations and species, such as C. specus (Adams et al. 2013). Acknowledgments We thank all the concerned citizens of the Nippenose Valley, especially David Hollick, for their encouragement, support, and friendship, without which this study could not have been completed. We also would like to thank James C. D. Lewis for collecting the samples. Joshua Gross provided the primers used in the study. The study was

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Espinasa et al. partially supported by the Cleveland Grotto Science Fund and the National Speleological Society Research Grant. Literature Cited Adams, G.L., Burr, B.M., Day, J.L., & Starkey, D.E. 2013. Cottus specus, a new troglomorphic species of sculpin (Cottidae) from southeastern Missouri. Zootaxa 3609: 484–494. Bilandžija, H., Cetković H., & Jeffery, W.R. 2012. Evolution of albinism in cave planthoppers by a convergent defect in the first step of melanin biosynthesis. Evolution and Development 14: 196–203. Espinasa, L., & Jeffery, W.R. 2003. A troglomorphic sculpin (Pisces: Cottidae) population: Geography, morphology and conservation status. Journal of Cave and Karst Studies 65: 93–100. Gross J.B., Borowsky, R., & Tabin, C.J. 2009. A Novel role for MC1R in the parallel evolution of depigmentation in independent populations of the cavefish Astyanax mexicanus. PLoS Genetics 5: e1000326. doi:10.1371/journal.pgen.1000326. Jeffery, W.R. 2001. Cavefish as a model system in evolutionary developmental biology. Developmental Biology 231: 1–12. Jeffery, W.R. 2009. Regressive evolution in Astyanax cavefish. Annual Review of Genetics 43: 25–47. Jeffery, W.R. 2010. Pleiotropy and eye degeneration in cavefish. Heredity 105: 495– 496. Protas, M.E., Hersey, C., Kochanek, D., Zhou, Y., Wilkens, H., Jeffery, W.R., Zon, L.I., Borowsky, R., & Tabin, C.J. 2006. Genetic analysis of cavefish reveals molecular convergence in the evolution of albinism. Nature Genetics 38: 107–111. Sadoglu, P.M.A. 1969. A second gene that affects eye and body color in Mexican blind cave fish. Journal of Heredity 60: 10–14. Schuldt, A., & Assmann, T. 2011. Belowground carabid beetle diversity in the western Palaearctic – effects of history and climate on range-restricted taxa (Coleoptera, Carabidae). Pp. 461–474 in Kotze, D.J., Assmann, T., Noordijk, J., Turin, H., & Vermeulen, R., eds. Carabid Beetles as Bioindicators: Biogeographical, Ecological and Environmental Studies. ZooKeys 100. doi: 10.3897/zookeys.100.1540

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