The teleost family Amblyopsidae (order Percopsiformes) comprises North America’s largest clade of stygobiotic (obligate cave-dwelling) fishes and has long been of interest to evolutionary biologists and ecologists studying adaptations to extreme subterranean habitats (reviewed in Niemiller and Poulson 2010). The taxonomy of Amblyopsidae has remained relative stable since the 1950s when Woods and Inger (1957) published a major taxonomic revision of the family, which included assigning Troglichthys rosae (Eigenmann 1898) to the genus Amblyopsis, recognizing Forbesichthys papilliferus (Forbes, 1882) as a junior synonym of Forbesichthys agassizii (Putnam, 1872), and recognizing Typhlichthys eigenmanni Charlton, 1933, Typhlichthys wyandotte Eigenmann, 1905 and Typhlichthys osborni Eigenmann, 1905 as junior synonyms of Typhlichthys subterraneus Girard, 1859. Cooper and Kuehne (1974) described the most recent amblyopsid to be recognized, Speoplatyrhinus poulsoni, a stygobiotic species known only from a single cave system in northern Alabama.

There has been a revival in amblyopsid systematics and taxonomy in recent years, as several studies have examined higher-level phylogenetic relationships as well as population level differentiation using molecular approaches (Niemiller and Fitzpatrick 2008, Dillman et al. 2011, Niemiller et al. 2012, 2013a, d). Several significant systematic revisions have been proposed, including the assignment of Amblyopsis rosae back into the genus Troglichthys (Niemiller et al. 2013a), resurrection of Forbesichthys papilliferus (Niemiller et al. 2013a) and resurrection of Typhlichthys eigenmanni (Niemiller et al. 2012) based on biogeographical and phylogenetic evidence. Eight species are currently recognized in Amblyopsidae, including three surface or primarily spring-dwelling taxa: Chologaster cornuta Agassiz 1853, Forbesichthys agassizii, and Forbesichthys papilliferus; and five obligate cave-dwelling taxa: Typhlichthys subterraneus, Typhlichthys eigenmanni, Troglichthys rosae, Speoplatyrhinus poulsoni and Amblyopsis spelaea DeKay 1842 (sensu Niemiller et al. 2013a, d). However, the multilocus phylogenetic study by Niemiller et al. (2012) also uncovered substantial cryptic genetic variation associated with hydrological boundaries in Typhlichthys subterraneus, suggesting that biodiversity is underestimated in Typhlichthys and perhaps other cavefish lineages. The recognition of such cryptic species has important implications for conservation and management (e.g., Niemiller and Fitzpatrick 2008, Niemiller et al. 2013b), but also for comparative ecological and evolutionary studies (e.g., Niemiller et al. 2013a, c).

A recent study of Amblyopsis spelaea based on phylogeographic structure of one mitochondrial and four nuclear loci identified two evolutionary lineages associated with the modern Ohio River (Niemiller et al. 2013d): a northern lineage located north of the Ohio River in Indiana and a southern lineage in Kentucky. Niemiller et al. (2013d) suggested that these two lineages might warrant recognition as distinct species (Fig. 1).

These northern and southern lineages were recovered as reciprocally monophyletic for two of the nuclear loci, s7 and rhodopsin, and the mtDNA locus nd2. Little variation was exhibited at the nuclear loci rag1 and tbr. The gene tree estimated from the mitochondrial nd2 locus contained two strongly supported clades (Bayesian posterior probability > 0.95) separated by 27 mutational steps with observed average uncorrected pairwise genetic distances of 3.1% between these lineages. Observed uncorrected pairwise genetic distances were considerably lower for all nuclear loci; however, segregating variation was observed in s7 and rhodopsin. A single nucleotide substitution at s7 segregated between the northern and southern lineages, whereas three nucleotide substitutions segregated at rhodopsin. The differences in rhodopsin include a mutation that results in a premature stop codon in the open reading frame of all individuals sampled in the southern lineage that is absent from the northern lineage.

The results of Niemiller et al. (2013d) strongly implicate the Ohio River as a significant barrier to dispersal and, consequently, an isolating mechanism facilitating divergence between populations located north and south of the river (Fig. 2). Poulson (1960) examined variation in morphology throughout much the northern distribution of Amblyopsis spelaea and found subtle differences in pigmentation and rudimentary eye size; however, he only examined specimens from the Mammoth Cave region for the southern range of the species. Therefore, it is unclear whether phenotypic differences exist between phylogenetic lineages identified by Niemiller et al. (2013d). In this study, we examined morphological variation from individuals of the northern and southern lineages of Amblyopsis identified by Niemiller et al. (2013d), and included specimens from populations for which material for DNA sequencing were previously unavailable. Based on our results, we describe the northern lineage as Amblyopsis hoosieri sp. n., from subterranean waters of southern Indiana.

Institutional abbreviations are as follows: CAS (California Academy of Science); INHS (Illinois Natural History Survey); IU (Indiana University); LSUMZ (Louisiana State University Museum of Natural Science); UMMZ (University of Michigan Museum of Zoology), YPM (Yale Peabody Museum). Non-type materials examined include: Amblyopsis spelaea: INHS 50129 (n=1), 60573 (n=1); UMMZ 146991 (n=1); 179149 (n=1); YPM 25294 (n=8, n=1 cleared and stained). Materials examined for the new species are listed below as types. Cleared and stained specimens were prepared following modifications of the method outlined by Taylor and Van Dyke (1985). Specimens were stained in Alcian blue for two days, then bleached in a potassium hydroxide solution, neutralized in a hydrogen peroxide + potassium hydroxide mixture, then transferred into trypsin to clear. Specimens were then placed in Alizarin red for 20 minutes and subsequently transferred to distilled water for 24 hours. Specimens were then placed back into trypsin for a final clearing and slowly moved up to full glycerin using a staggered glycerin/potassium hydroxide mixture that slowly increased glycerin in 10% intervals up to 100% over a period of a week.

Radiographs were made for all specimens using a Faxitron x-ray cabinet. All meristics (numbers of fin rays and vertebrae) were counted using these radiographs.

Morphometric measurements, following Hubbs and Lagler (2004), were recorded to the nearest 0.1 mm using digital calipers and included: standard length (SL), head length (HL), head width, upper jaw length, body depth (depth of body at deepest point), pectoral-fin length, caudal-fin length, pelvic-fin length, dorsal-fin base, anal-fin base, caudal peduncle length, caudal peduncle width, caudal peduncle depth, predorsal length, prepelvic length, and preanal length. Other traditional measurements, such as, snout length, interorbital width and orbit diameter are excluded because of the absence of eyes (externally) in these taxa. An additional measurement, body width, was taken directly posterior to the opercula on the widest part of the body. We also conducted an analysis of covariance (ANCOVA) to compare body width and body depth among species with standard length as a covariate in R (v3.0.2; R Development Core Team 2013).

Forty-one specimens were examined, 30 from north of the Ohio River in Indiana, and 11 from south of the river in Kentucky. Specimens from Kentucky included the type locality of Amblyopsis spelaea, Mammoth Cave in Edmonson County. Figure 3 (A–C) shows the results of analyses of covariance (ANCOVA) comparing body depth and body width versus SL. Notably, Amblyopsis hoosieri has a deeper body compared to Amblyopsis spelaea (ANCOVA, p < 0.001; Fig. 3A, C). The body is also wider in Amblyopsis hoosieri compared to Amblyopsis spelaea (ANCOVA, p < 0.001; Fig. 3B, C). Except for juveniles (those under 50 mm), individuals of the new species are deeper and wider bodied than individuals of Amblyopsis spelaea (Fig. 3C). Given individuals of the same standard length, one would expect those of the new species to be much more robust.

We describe a new species of North American cavefish, Amblyopsis hoosieri, that is distinguished from its sole congener Amblyopsis spelaea based on body and pectoral-fin shape and the absence of a stop codon in rhodopsin among other molecular and morphological features. In addition, the distributions of the two species of Amblyopsis are separated by the Ohio River, which has downcut through major cave-bearing rock strata and has subsequently isolated populations on the north (Amblyopsis hoosieri) and south (Amblyopsis spelaea) sides of the river. Cavefish diversity is certainly underestimated globally but perhaps particularly in North America: this is the first new cavefish species from the U.S. in 40 years.

Notably, previous cavefish researchers did not recognize this taxon as novel. Eigenmann (1899) noted that some individuals of Amblyopsis had more developed cones than others and Poulson (1960) noted qualitatively greater non-external pigmentation and degenerate “eye” size of specimens of Amblyopsis spelaea from south of the Ohio River. Unfortunately, due to the limited number of specimens available we were not able to fully examine these internal features. However, we found that there are several lines of evidence to distinguish the two species based on external morphological features, molecular data and geography.

Eigenmann (1905) described Typhlichthys wyandotte from “north of the Ohio River, from a well near Corydon, Indiana.” The type locality is located within the distribution of Amblyopsis hoosieri and well outside the known distribution of Typhlichthys in the Interior Plateau, which ranges from northern Alabama and northwestern Georgia through central Tennessee into south-central Kentucky (Mammoth Cave region). The type locality was believed to be destroyed (Woods and Inger 1957), although a well-like entrance into a cave has been located in Corydon, Indiana, and may be the type locality (Black, pers. comm. in Lewis 2002b). Unfortunately, the only known specimen (the holotype; formerly IU 4646, currently CAS 91988) is very badly damaged and in several pieces, with most of the head lost. In his description, Eigenmann (1905) stated that this species is more slender than Typhlichthys from south of the Ohio River. This condition is opposite of the situation in Amblyopsis, where individuals north of Ohio River are less slender than those south of the river. A survey of 200+ caves in the same drainage basin as the possible type locality has only documented Amblyopsis and has failed to find Typhlichthys (Lewis 1998). Typhlichthys wyandotte is currently considered a junior synonym of Typhlichthys subterraneus (Woods and Inger 1957), but it is unclear whether the poorly preserved holotype is a member of Typhlichthys or Amblyopsis.

Molecular data have become an important tool to help identify cryptic or otherwise poorly recognized species level diversity, particularly among subterranean taxa (Chakrabarty 2010, Chakrabarty et al. 2012, Sparks and Chakrabarty 2012). Generally lacking eyes, pigmentation, and other common features of sighted organisms, subterranean fish species have few diagnostic features. Molecular data have been used to discover and diagnose cavefish diversity only recently, but these data are powerful and can surely help increase our understanding of this poorly studied fauna.

Conservation status. Amblyopsis spelaea, and Amblyopsis hoosieri by extension, is considered endangered in Indiana because of presumed vulnerability to groundwater pollution and other perturbations of aquatic subterranean habitats. The species is considered “Endangered” (S1) in Indiana by NatureServe (2013) because of the few occurrences of occurrences, small population sizes and being restricted to subterranean habitats that are highly vulnerable to anthropogenic activities. Amblyopsis spelaea is considered “Vulnerable” on the IUCN Red List (Gimenez Dixon 1996). Amblyopsis hoosieri should have the same threat category at minimum or be at greater risk of extinction. Amblyopsis hoosieri is known from at least 74 localities, but most localities appear to represent sink rather source populations (Pearson and Boston 1995, McCandless 2005). However, a few cave systems contain large populations based on direct counts during visual encounter surveys that likely are source populations, including Eric’s River Cave in Crawford Co., and Blue Spring Caverns, Donaldson Cave and Upper Twin Cave, Lawrence Co. (Pearson and Boston 1995, McCandless 2005, Niemiller and Poulson 2010).

Potential threats to populations of Amblyopsis hoosieri are discussed in detail (for Amblyopsis spelaea) by Keith (1988), Pearson and Boston (1995), Lewis (2002a) and Niemiller and Poulson (2010). These threats include sedimentation related to agriculture, increased human visitation and collection, and groundwater pollution, particularly from pesticide, herbicide and fertilizer use. Some localities have been directly impacted by anthropogenic activities. Keith (1988) reported that two blind cavefish localities in Indiana were either partially or completely destroyed by quarrying. Groundwater contamination from pesticides was attributed to the cause of “broken-back syndrome” in the population at Donaldson Cave, Lawrence County (Keith and Poulson 1981). At least two populations are indirectly affected by commercial cave tours in Lawrence County (Pearson and Boston 1995; Niemiller and Poulson 2010). Over-collection for scientific studies during the late 1800s and early 1900s may have impacted some populations in Lawrence County. Dozens to hundreds of cavefish were collected from the “Mitchell Caves” (Bronson-Donaldson and Twin Cave systems) by Eigenmann, Payne and others (e.g., Eigenmann 1899, 1903, 1909; Ramsey 1901, Payne 1907) for experiments on cave adaptation. These caves are now protected and located within Spring Mill State Park. The state of Indiana has implemented measures to help protect populations of Amblyopsis, including restricting access to caves and regulating recreational activities permitted. However, the delineating of drainage basins and potential sources of contamination as well as protection of surface and subsurface drainage basins is probably the most important conservation measure to protect the species (Niemiller and Poulson 2010).