Espinasa & Espinasa-Pereña Sistema Huautla: Deep cave exploration and the opportunity for new molecular studies of its fauna Luis Espinasa1 & Ramón Espinasa-Pereña2 1 School of Science, Marist College, 3399 North Rd, Poughkeepsie, New York 12601, USA [email protected] (corresponding author) 2 Subdirección de Riesgo Geológico. Centro Nacional de Prevención de Desastres, México Key Words: Anelpistina specusprofundi, Anelpistina asymmetrica, Cubacubana, Cubacubaninae, Nicoletiidae, Zygentoma, Insecta, Thysanura, Silverfish, scorpion Alacran tartarus, troglobite, Huautla, Sótano de San Agustín, Oaxaca, México. From 1967 to 1994, the vast underground passages of the Sistema Huautla in Oaxaca, México, received regular visits by cave explorers. Thanks to the effort of cavers such as Andy Grubbs and Allan Cobb, we now know that the Huautla system is inhabited by at least ten troglobionts (Stelle and Smith 2005). Regrettably, there has not been a major expedition to this cave in almost 20 years. The last major exploration occurred in 1994 when an international team of cavers and divers established Sistema Huautla as the deepest cave in the Western Hemisphere but were halted by a large sump (Sump 9) at a depth of 1,475 m (Stone et al. 2002). At depths of over 750 m below the nearest cave entrance, the food chain includes predators such as the troglobiotic scorpion Alacran tartarus Francke, 1982, and the tarantula Spelopelma grieta Gertsch, 1982. The community of organisms also includes the rather long (90 mm including antennae and caudal appendages) nicoletiid silverfish Anelpistina specusprofundi (Insecta: Zygentoma: Cubacubaninae), described by Espinasa and Vuong (2008). In their 2008 study, Espinasa and Vuong posited that A. specusprofundi is the sister species of the troglobiotic A. asymmetrica Espinasa, 2000, because of their morphological similarity. Unfortunately, with no fresh samples from which DNA evidence could be obtained, this hypothesis has not been substantiated. Anelpistina asymmetrica inhabits three caves in Puebla and Oaxaca (Espinasa 2000; Espinasa et al. 2007), which are about 30 km from Sistema Huautla. Females of both species are almost undistinguishable. Males are more easily differentiated based on their sexual secondary characters because similarly sized adult males of A. specusprofundi lack the curved cerci and the asymmetric pedicellus of adult A. asymmetrica males. The clearest character allowing for differentiation between the two species is that although both have long and slender bodies and appendages, as expected from troglobiotic silverfish, A. asymmetrica appears to be proportionally longer 2014 Speleobiology Notes 6: 62–67 62 Espinasa & Espinasa-Pereña and more slender than A. specusprofundi (Espinasa and Vuong 2008). If they are indeed closely related, an interesting possibility is that they share a common ancestor who was already adapted to caves. Both species are highly troglomorphic, adapted to survive with the minimal resources found in deep caves, and they inhabit caves in contiguous karstic mountain ranges. Biological studies of the deepest caves in the world are hampered by the extreme difficulty of reaching these remote habitats. Sampling is typically possible only within organized expeditions (Sendra and Reboleira 2012). Since 1994, opportunities to further understand Sistema Huautla’s troglobiotic community were nonexistent. With just a handful of specimens collected in the 1980s housed at various museums and preserved under conditions that do not allow for easy DNA amplification and sequencing, molecular studies could not be done. An opportunity to obtain fresh samples finally arose with the 2013 British-lead expedition, with a strong support team of more than 40 cavers from the United Kingdom, Canada, United States, and Mexico. The expedition leader, Chris Jewel, was contacted by the authors of this manuscript and an agreement was reached wherein several of the expedition’s members, including one of us (RE), volunteered to dedicate time and effort to collecting specimens of interest. The purpose of this study was to document a particular behavior of the troglobiotic scorpion Alacran tartarus inhabiting this deep cave and collect new samples of A. specusprofundi to better understand their morphology and sequence their DNA. Fieldwork in Sistema Huautla from February through April 2013 produced observations of three individuals of the troglobiotic scorpion, with no vouchers collected. Previous reports had indicated that the scorpions could be found underwater (A. Grubbs pers. comm.) and this was confirmed during the 2013 expedition. Out of three Alacran tartarus observations, one was reported underwater in a gour (rimstone) pool near Camp 3 and another individual was seen underwater in Sump 9 at a depth of 6 meters (Jewell 2014). The second individual responded to the diver’s gloved hand by arching its body in an apparently defensive or aggressive manner, suggesting that it was functioning normally underwater (A. Walmsley pers. comm.). It appears that this troglobiotic scorpion may truly be adapted for living extended periods of time underwater. Based on our literature search, A. tartarus represents one of the few, if not the only, scorpion in the world that is routinely found underwater. Three A. specusprofundi were collected (Figure 1): a juvenile female (12 mm long), an adult female (16 mm) and a male (13 mm). The specimens were found crawling near Camp 3 in the Metro section of the cave at a depth of close to 750 m below the entrance (Figure 2). Approximately 10 individuals were observed in this area, but reports indicate that there where many more between Sump 1 and Sump 9. 2014 Speleobiology Notes 6: 62–67 63 Espinasa & Espinasa-Pereña Figure 1. Three new specimens of Anelpistina specusprofundi collected during the 2013 expedition to Sistema Huautla, Oaxaca, México. From left to right, juvenile female 12 mm, male 13 mm, and female 16 mm. Previous analyses of postembryonic development of A. specusprofundi were based on a limited number of specimens (Espinasa and Vuong 2008). That study showed that in a 10 mm juvenile female, the ovipositor was just beginning to develop, barely reaching the base of stylets IX, and the gonapophyses was without distinct annuli. The other female specimens from the 2008 study were 15–19 mm of body length and had ovipositors surpassing stylets IX by about 1/2 the length of the stylets, while the gonapophyses had approximately 18 annuli. Due to the large gap in size of the available specimens, it was unclear at what length adult female morphology is achieved. With the 2013 collection of a 12 mm long juvenile female, it is now clear that by this size the ovipositor has already achieved adult proportions. Genomic DNA from the juvenile female was extracted using Qiagen’s DNEasy® Tissue Kit by digesting a leg in lysis buffer. Amplification and sequencing of a 515 base- pair (bp) fragment of the 16S rRNA mitochondrial gene followed standard protocols and primers for the 16S rRNA fragment used in the past for nicoletiids (Espinasa and Giribet 2009). Chromatograms obtained from the automated sequencer were read and contigs made using the sequence editing software SequencherTM 3.0. External primers were excluded from the analyses. The sequence of A. specusprofundi was accessioned into GenBank (GenBank no. KJ128287). The Basic Local Alignment Search Tool (BLAST) was used to compare our query sequence with available nicoletiid GenBank sequences. 2014 Speleobiology Notes 6: 62–67 64 Espinasa & Espinasa-Pereña Figure 2. Profile and plan view maps of Sistema Huautla, Oaxaca, México, the deepest cave in the Western Hemisphere. Arrows point to the location where nicoletiid silverfish were collected. BLAST analysis showed A. specusprofundi to be more similar to A. asymmetrica (GenBank no. DQ280128) than to any other nicoletiid insect that has had its 16S sequenced. In their overlapping sequence (only a fragment of 463 bp has been sequenced in A. asymmetrica), they differ by 60 bp (12.9%). Espinasa and Giribet 2014 Speleobiology Notes 6: 62–67 65 Espinasa & Espinasa-Pereña (2009) have used this particular 16S rRNA fragment as a reference for recognizing species within nicoletiids. They observed that pairs of specimens within a population of species examined differ by an average of 1.7 nucleotides (0.4%; range 0–7 nucleotides; n=29), by 3.4 nucleotides (0.7%; range 0–13 nucleotides; n=22) in different populations of the same species, and by 31.2 nucleotides (6.7%; range 10–64 nucleotides; n=14) among sister species. The difference of at least 60 bp between A. specusprofundi and A. asymmetrica supports what was proposed by Espinasa and Vuong (2008) based on morphology; that A. specusprofundi is a different species, but among the described nicoletiids, they are most closely related to A. asymmetrica. A 60 bp difference is equivalent to approximately 0.5–5 million years since A. specusprofundi and A. asymmetrica shared a common ancestor, based on molecular clock calibrations in other nicolettids (Espinasa et al. 2011). When two closely related troglobiotic species inhabit caves 30 km apart in contiguous karstic mountain ranges, a parsimonious hypothesis is that they shared a common ancestor, which was already cave adapted. The large time available of up to millions of years allows for great erosional changes in the region and the development of cave systems spanning the sections in-between the two current karstic areas. Those systems would have since eroded away. While we certainly do not suggest that there has ever been a continuous cave connecting both areas, we propose that through current and extant systems of caves, interstices, and the epikarst, with the long time range available, the descendants of this ancestral population were able to migrate underground between the two areas. An alternative, but less parsimonious possibility is that a common surface ancestor colonized both karstic regions independently. Regardless of which scenario is correct, it appears that A. specusprofundi and A. asymmetrica have a long evolutionary history in subterranean habitat. Acknowledgments We thank all members of the Huautla 2013 cave diving expedition.