Weck & Taylor

Life history studies of a cave-dwelling population of Physa snails (: Basommatophora: ) from southwestern Illinois

Robert G. Weck1 & Steven J. Taylor2

1Biology Department, Southwestern Illinois College, 2500 Carlyle Ave, Belleville, Illinois, 62221, USA [email protected] (corresponding author)

2Illinois Natural History Survey, Prairie Research Institute, University of Illinois at Urbana-Champaign, 1816 S. Oak St., Champaign, Illinois, 61820, USA [email protected]

Key Words: albinism, Salem Plateau, growth rates, fecundity, pigmentation, , Fogelpole Cave, Monroe County.

Physid snails are a common part of the aquatic community in caves of the Salem Plateau in southwestern Illinois, USA. In spite of their widespread occurrence, the life histories of cave-dwelling physids are essentially unknown, and most populations have not been positively identified using current classification schemes. Hubricht (1950) suggested that the Physa populations in caves of the Ozarks are derived from epigean snails washed into caves and exist as troglophiles. This seems to be the case for the Physa acuta Draparnaud, 1805 population in Stemler Cave, Illinois (Weck and Mafla- Mills in preparation). No obligate cave-adapted physid species is known from Illinois. Indeed, the only well-documented troglobitic physid is Physa spelunca Turner & Clench, 1974, a single-cave endemic from Lower Kane Cave, Wyoming, USA. Turner and Clench (1974) described several troglomorphic characters in the series of P. spelunca they examined, including reduced eyes, lack of pigmentation, large protoconch, and small adult body size. Porter et al. (2002) reported that P. spelunca population densities were extremely high and contained red and black color morphs. The species is phylogenetically distinct based on mitochondrial DNA analysis (Wethington and Lydeard 2007).

Fogelpole Cave, in Monroe County, is Illinois' largest and most biodiverse cave with over 24 kilometers of known passage and 14 troglobitic species (Lewis et al. 2003). Peck and Lewis (1978) noted that the physid snails in Fogelpole Cave were depigmented and may represent a troglobitic form of Physa halei Lea, 1864. Based on the large size of mature adults raised in captivity (19 mm shell length) and examination of photographs of adults by Robert T. Dillon (personal communication, 19 September 2014), the Fogelpole Cave populations have been also identified as Physa gyrina Say, 1821. An ongoing phylogeographic study of cave-dwelling physids from the Salem

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Plateau of southwestern Illinois by our research group is designed to determine the identity of this population (Niemiller et al. in preparation). Previously we observed that pigmentation in the Fogelpole Cave population is highly polymorphic (Figure 1). Depigmentation is a common troglomorphic feature in obligate cave (Culver 2005).

Figure 1. Lab raised pigmented (top) and depigmented (bottom) morphs of Physa sp. from Fogelpole Cave, Monroe County, Illinois. Photographs by Matthew L. Niemiller.

A troglomorphy (T) scale was created to classify pigmentation phenotypes (Figure 2). The scale ranges from full pigmentation in shell, mantle, and body (T1) to a complete lack of dark pigment in all parts of the (T5).

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Objectives of the studies reported here were to: (1) determine the pattern of inheritance of pigment by conducting controlled crosses; (2) compare fecundity of pigment phenotypes under simulated cave conditions; (3) quantify the frequency of each phenotype of snails in Fogelpole Cave; and (4) compare growth rates of offspring from different pigment morphs.

T1 T2 T3 T4 T5

Figure 2. Troglomorphy scale used to classify pigmentation phenotypes in the Fogelpole Cave, Monroe County, Illinois population of Physa sp. Drawings by Robert G. Weck.

Inheritance of pigmentation

Snails collected from Fogelpole Cave on 10 April 2014 were used to establish true- breeding lab strains of the T1 and T5 phenotypes. Depigmented snails always produced T4 or T5 offspring, but pigmented snails produced offspring across the full pigment spectrum (T1–T5) and were inbred through several generations to create a true breeding T1 line. Parental generation crosses between T1 and T5 lines were established by pairing immature snails (ca. 2 mm shell length) in plastic cups of groundwater. All animals were fed ground algae-based fish food. Upon maturation and egg production by parent snails, F1 embryos were isolated. Physids are hermaphrodites and capable of self-fertilization, though they typically outcross when mates are available (Dillon and Wethington 1992). To ensure that F1 offspring were the result of outcrossing, the depigmented parents from each cross were periodically isolated and allowed to deposit eggs. Individual clutches of embryos were screened for pigmentation prior to hatching. Since the snails came from true breeding cultures, any pigmented embryo produced by a depigmented adult must be a hybrid. Hybrid F1 snails (from clutches containing only pigmented embryos) from 13 different crosses were isolated and reared to maturity. Forty-three clutches of F2 embryos, containing 984 offspring were scored. The presence of black pigment in the eyes of late stage embryos was used to determine the pigmented phenotype (Figure 3).

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Figure 3. Pigmented (dark eyes) and depigmented (black arrow) F2 Physa sp. embryos from Fogelpole Cave, Monroe County, Illinois. Photograph by Robert G. Weck.

Chi square analysis (Table 1) strongly supports the hypothesis that F2 offspring segregate in a 3:1 pigmented to depigmented ratio as predicted if the depigmented phenotype represents albinism due to a recessive allele at a single genetic locus. Snails with the T4 and T5 phenotype are homozygous recessive and thus may be considered albino. However, the spectrum of pigment phenotypes seen in the population (Figures 1 & 2) suggests overall pigmentation is a polygenic trait. Alleles at multiple additional loci likely control amount and distribution of melanin in pigmented morphs.

Table 1. Chi square analysis comparing observed and expected frequency of pigmented and depigmented phenotypes in F2 Physa sp. embryos from Fogelpole Cave, Monroe County, Illinois.

Expected Deviation Phenotype Observed d2 d2/e (e) (d) Pigmented 735 738 -3 9 0.012

Depigmented 249 246 3 9 0.037

Σd2/e= 0.049

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Population structure

Quadrat sampling was used to determine the distribution of each phenotype class in a section of mainstream Fogelpole Cave on 26 October 2015. A series of eight quadrat samples (0.42 m2) were censused in four stream reaches included pool and riffle habitats. A total of 57 Physa sp. snails was observed. The average density is estimated at 16.79 snails/m2. Over 67% of the population sampled showed marked depigmentation (e.g., T4 or T5 phenotypes) (Figure 4). No T1 morphs were observed and the majority (73.7%) of the pigmented snails were T3 morphs. Snail shell length ranged from 1 mm to 8 mm, (mean 4.8 mm). Pigmented (T2 or T3) snails were larger than depigmented snails, but the sample size was small and the differences were not significant (Figure 4).

Figure 4. Shell length and distribution of Physa sp. snail pigmentation phenotypes in mainstream Fogelpole Cave, Monroe County, Illinois. Error bars represent ± one standard deviation.

Fecundity

Seven wild caught snails were sorted by similar phenotype (T1–T2; n=2), (T3; n=2), (T4–T5; n=3), held in the laboratory under simulated cave conditions (dark cooler set at 14 oC) in plastic cups of groundwater and fed ground algae-based fish food ad libitum. Fecundity was measured by counting all eggs produced for a 30-day period between 30 May and 29 June 2014. Data are reported in mean number of eggs/snail/day. Darkly pigmented snails produced nearly six times more eggs per day than T3 or depigmented (T4–T5) snails (Figure 5).

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Figure 5. Mean number of eggs produced per snail per day for three phenotype groups of parental Physa sp. snails from Fogelpole Cave, Monroe County, Illinois.

Growth rates

Several clutches of embryos were isolated from each of the phenotype pairings used in the fecundity study (T1–T2; T3; T4–T5) and allowed to hatch. Twenty circa 1.5mm hatchlings from each set were isolated in separate deep Petri dishes of groundwater. Ten snails were reared under simulated surface conditions (ca. 20–25 oC, 12 hrs light/day) and 10 under simulated cave conditions (dark cooler set at 14 oC). All snails were fed ground algae-based fish food ad libitum. Shell length of each snail was measured with a millimeter ruler twice a week for 6.5 weeks between 4 June and 21 July 2014, by examining specimens under a dissecting microscope. In all treatments, snails exhibited faster initial growth rates in surface conditions than in simulated cave conditions, though only offspring of depigmented (T4–T5) parents actually grew to a larger size in surface conditions than in simulated cave conditions (Figure 6). Offspring of T1–T2 in surface conditions experienced a plateau in growth after four weeks, presumably shifting energy to egg production as the first egg masses were produced at this point of inflection in the growth curve. This pattern was observed also in recent growth studies of a cave-dwelling population of Physa acuta from southwestern Illinois (Weck and Mafla-Mills in preparation). The first egg production in offspring of T4–T5 in surface conditions was seven days later. No snails produced eggs under simulated cave treatments during the 6.5 week study period, but a single individual in the T1–T2 group deposited a clutch of eggs on 23 July 2014, one day after data collection ceased. The overall patterns seen in offspring of T3 snails was roughly intermediate.

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Figure 6. Growth rates of offspring Fogelpole Cave, Monroe County, Illinois snails from T1–T2 parents (a), T3 parents (b), and T4–T5 parents (c) under surface (n=10/snail morph) and cave (n=10/snail morph) conditions. Black arrows indicate first instance of egg production. Error bars represent ± one standard deviation.

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In this study, a reduction in number of eggs produced per unit time, a delayed onset of reproduction and slower growth rates were observed in depigmented (T4–T5) snails. These life history attributes are well-documented phenomena in cave-adapted animals (Culver 2005). In our laboratory study, offspring of both T1–T2 and T3 pigmented parents raised under simulated cave conditions and provided food ad libitium grew to a size exceeding the maximum snail length observed in Fogelpole Cave. Depigmented snails kept in the lab well beyond the end of the study eventually reached shell lengths of 14.5 mm, though none grew as large as pigmented specimens, some of which reached 19 mm shell length. The life history data presented in this study, and the ease with which these animals are maintained in the laboratory, suggest that Physa sp. in Fogelpole Cave could make a good model for the study of evolutionary adaptations to subterranean life.

Pigmented snails may be recent introductions from surface habitats that interbreed with resident depigmented, possibly troglomorphic individuals. Indeed, it is common to encounter long-eared sunfish, yellow bullheads, and other surface pond fauna in the main stream of Fogelpole Cave, suggesting regular connectivity with surface waters. Ongoing genetic and morphometric studies by our research group (Niemiller et al. in preparation) should shed more light on the biology of subterranean physid snails of Illinois' Salem Plateau.

Acknowledgments

Stephanie Mafla-Mills (University of Missouri, St. Louis, Missouri, USA) and Abigail Ragsdale (University of California, Santa Cruz, California, USA) helped collect data and care for snails in the lab. Robert T. Dillon (College of Charleston, Charleston, South Carolina, USA) and Charles Lydeard (Western Illinois University, Macomb, Illinois, USA) provided advice on snail culture techniques and Dr. Dillon identified snails on the basis of photographs. Students in the spring 2014 genetics class at Southwestern Illinois College (Belleville, Illinois, USA) assisted in creating true breeding line of T1 and T5 snails used in the genetic crosses. We thank Matthew L. Niemiller (University of Illinois at Urbana-Champaign, Champaign, Illinois, USA) for allowing use of his photographs and Patrick Weck (Columbia, Illinois, USA) for assistance in creating the drawings of the pigmentation troglomorphy scale. Fieldwork was conducted under an Illinois Nature Preserves Commission permit to Clifftop NFP (Maeystown, Illinois, USA), and we are grateful for Clifftop for allowing us to work underground in Paul Wightman Subterranean Nature Preserve.

Literature Cited

Culver, D. 2005. Life history evolution. Pp. 346–349 in Culver, D. & White. W., eds. Encyclopedia of Caves. Elsevier. Amsterdam, Netherlands.

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Dillon, R.T., & Wethington, A.R. 1992. The inheritance of albinism in a , Physa heterostropha. Journal of Heredity 83: 208–210.

Hubricht, L. 1950. The invertebrate fauna of Ozark caves. The National Speleological Society Bulletin 12: 16–17.

Lewis, J., Moss, P., Tecic, D., & Nelson, M. 2003. A conservation focused inventory of subterranean invertebrates of the Southwestern Illinois Karst. Journal of Cave and Karst Studies 65: 9–21.

Peck, S., & Lewis, J. 1978. Zoogeography and evolution of the subterranean invertebrate faunas of Illinois and southeastern Missouri. The National Speleological Society Bulletin 40: 39–58.

Porter, M.L., Russell S., Engel A.S., & Stern L.A. 2002. Population studies of the endemic snail Physa spelunca (Gastropoda: Physidae) from Lower Kane Cave, Wyoming. Journal of Cave and Karst Studies 64: 181.

Turner, R.D., & Clench, W.J. 1974. A new blind Physa from Wyoming with notes on its adaptation to the cave environment. Nautilus 88: 80–85.

Wethington, A.R., & Lydeard, C. 2007. A molecular phylogeny of Physidae (Gastropoda: Basommatophora) based on mitochondrial DNA sequences. Journal of Molluscan Studies 73: 241–257.

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