Espinasa, Christoforides & Morfessis Sequence analyses of the 16S rRNA of epigean and hypogean diplurans in the Jumandi Cave area, Ecuador Luis Espinasa1,2, Sara Christoforides1 & Stella E. Morfessis1 1 School of Science, Marist College, 3399 North Rd, Poughkeepsie, New York 12601, USA 2 [email protected] (corresponding author) Key Words: Diplura, Jumandi Cave, Ecuador, troglophile, troglobite, Arthropoda, Insecta, surface, cave, depigmentation, eyeless, Nicoletiidae. One of the most visited caves in Ecuador is Jumandi Cave (0o 52.5028’ S, 77o 47.5587’ W). Jumandi Cave is 660 meters above sea level, 4 kilometers north of the village of Archidona, and 14 km north of the town of Tena in the Napo Province (Peck 1985). The cave is a single stream passage with side corridors. For a full description of the cave and a map see Peck (1985). Temperature inside the cave is 22 °C (Peck 1985). Bats and bat guano is scarce throughout the cave. A stream takes up most of the cave floor and brings in considerable plant debris that may be the food source for the aquatic invertebrate and vertebrate community. The most distinguished inhabitant of this cave is the endemic cave-adapted catfish, Astroblepus pholeter (Collette, 1962) (Collette 1962; Haspel et al. 2012). Stewart Peck, professor and entomologist at the University of Carleton, visited Jumandi Cave in 1984 and conducted a bioinventory. He reported the presence of 22 species of invertebrates as well as the Astroblepus catfish (Peck 1985). According to Peck, 19 species of the invertebrates were troglophiles and three were trogloxenes. The only reported troglobiont was Astroblepus pholeter (Peck 1985). Among the reported troglophiles were two female diplurans, family Campodeidae (Insecta), found on the silt banks in the cave. These specimens had similarities with the endogean species Plusiocampa (Litocampa) brasiliensis Wygozinsky, 1944, but Peck considered them to be an undescribed troglophilic species of Plusiocampa (Litocampa). On 25 December 2015, Jumandi Cave was visited by one of us (LE). Multiple diplurans were observed on the mud banks of a side gallery. Specimens were seen openly walking on the surface of the substrate, and none were seen underneath rocks, although no exhaustive search was conducted. All specimens observed had long appendages; their antennae were as long as their bodies, and their cerci were estimated as twice as long. As they walked, the diplurans moved their cerci side to side. On that same date, a small bioinventory of the surface fauna residing under rocks and logs was performed. Diplurans were found to be abundant in this environment just 50 m from the entrance of Jumandi Cave. These surface diplurans appeared to be more robust and had shorter appendages. In particular, their cerci were estimated to be about the same length as their bodies 2017 Speleobiology Notes 9: 18–22 18 Espinasa, Christoforides & Morfessis (Figure 1). Regrettably, cerci are quite fragile and broke during the collection of specimens, and no direct measurement could be obtained. Figure 1. A surface dipluran found under rocks and logs just 50 m from the entrance of Jumandi Cave (A), and a dipluran collected from inside the cave (B). Notice that the cavernicole specimen is proportionally slenderer. Cerci and antennae are quite fragile and broke off during collection. In live specimens, the cerci of cave specimens are longer than the body, while in surface specimens, cerci are barely the size of their body. According to Trajano (2012), “Troglophiles are able to complete their life cycles both in the hypogean and in the epigean environment, forming populations in both habitats, with individuals commuting between them and maintaining genetic flow between these populations”. Since, according to Peck (1985), the cave diplurans were troglophiles, the following hypothesis could be formulated: surface diplurans living under rocks just meters away from the cave entrance belong to the same species as the specimens inside the cave. Our observation of their morphology did not fully support this hypothesis; however, tail length of a fragile organism is weak evidence as their tail can easily break off. The purpose of this study was to sequence DNA from two cave and two surface diplurans to see how closely related they were to each other. 2017 Speleobiology Notes 9: 18–22 19 Espinasa, Christoforides & Morfessis Genomic DNA was extracted using Qiagen’s DNEasy® Tissue Kit by digesting a leg in lysis buffer. Amplification and sequencing of the 16S rRNA fragment was done as in Espinasa and Giribet (2009). Amplification was carried out with the 16Sar and 16Sb primers for 16S rRNA in a 50 µl volume reaction using the Qiagen Multiplex PCR kit. The PCR program consisted of an initial denaturing step at 94 ºC for 60 sec, 35 amplification cycles (94 ºC for 15 sec, 49 ºC for 15 sec, 72 ºC for 15 sec), and a final step at 72 ºC for 6 min. Chromatograms obtained from the automated sequencer were read and contigs constructed using the sequence editing software SequencherTM 3.0. External primers were excluded from the analyses. The Basic Local Alignment Search Tool (BLAST) was used to compare our query sequence with available GenBank sequences. Sequences were aligned using ClustalW. The 16S rRNA fragment of both surface specimens was identical and 455 bp in length (GenBank no. KX987125). The two cave specimens also had an identical 16S rRNA fragment but was 464 bp long (GenBank no. KX987124). Alignment between the four specimens showed that the cave and the surface population were very divergent. They differed by 79 bp (17%), including 19 insertions or deletions (Figure 2). When compared against other available dipluran sequences in GenBank, the surface specimens were most similar to Campodea lubbocki Silvestri, 1912 (GenBank no. DQ529237), yet still differed by 21.0%. The cave specimens were most similar to Campodea sp. (GenBank no. JN615251) but differed by 20.5%. Figure 2. Partial sequence of the 16S rRNA of surface (first sequence) and cave (second) diplurans. Chromatograms are shown below. Their sequences are considerably different (17%), including multiple insertions and deletions, represented here by blue colons. Currently, there are only seven 16S rRNA sequences reported for diplurans of the family Campodeidae in GenBank. They are distributed among three different species, and none from members of the genus Litocampa, to which Peck (1985) assigned the Jumandi 2017 Speleobiology Notes 9: 18–22 20 Espinasa, Christoforides & Morfessis Cave specimens. As such, it is difficult to calibrate a molecular clock for this group or delineate with confidence how many base pair differences support that two populations belong to different species. The 16S rRNA fragment examined in this study has been used in other Apterygota insects. Hundreds of 16S rRNA sequences have been analyzed for nicoletiid species across the subfamily Cubacubaninae (Espinasa et al. 2015). For this group, within-population genetic variation averages 0.3% and 0.7% among populations of the same species. Divergence among sister species averages 6.2%. Since it is unlikely that the two Apterygota insects would have a molecular clock over ten times faster than each other, it is highly likely that the surface and cave specimens collected near the entrance and within Jumandi Cave are different species. In conclusion, our DNA data support recognition of the cave diplurans in Jumandi Cave as a different species from the surface specimens outside the cave, despite being collected just 50 m away from each other. An intriguing possibility is that the cave population is actually troglobiotic and endemic to the subterranean environment. While subjective, behavioral observations showed that the specimens in the cave were openly walking on the surface of the substrate, unlike small arthropods that live on the surface and are constantly being preyed upon, and tend to hide under rocks. Cave-adapted organisms typically have reduced predation pressures and thus show reduced prey avoidance behaviors (Plath and Schlupp 2008). It is unlikely that the specimens observed by us in the cave could survive for long in surface habitats if they did not seek refuge under rocks and logs. While the study was not designed with a taxonomic approach, the little morphological data available also support that the cave population may be cave adapted. Their slender bodies and extremely long cerci that they move side to side could be a feature of troglomorphy. Such long cerci were not found in any of the surface specimens examined. We can only suggest at this time that these two groups of diplurans are different species and that the cave group is troglobiotic; this hypothesis will require further testing and verification. Acknowledgments We thank Jenna Robinson, Anthony Finocchiaro, and Joseph Kopp, members of the Jumandi 2016 cave expedition, for their help collecting specimens. Luis Alberto Chacha Guayña provided the collecting permit and support while exploring the cave. Students of the BIOL320-112 Genetics Spring 2016 course at Marist College performed DNA sequencing. The School of Science at Marist College gave partial support for the publication of this article. Literature Cited Collette, B.B. 1962. Astroblepus pholeter, a new species of cave dwelling catfish from Eastern Ecuador. Proceedings of the Biological Society of Washington 75: 311–314. Espinasa, L., Bartolo, N.D., & Sloat, S.A. 2015. A new epigean species of the genus Anelpistina (Insecta: Zygentoma: Nicoletiidae) from Sierra de El Abra, Taninul, Mexico. European Journal of Taxonomy 156: 1–7. 2017 Speleobiology Notes 9: 18–22 21 Espinasa, Christoforides & Morfessis Espinasa, L., & Giribet, G. 2009. Living in the dark—species delimitation based on combined molecular and morphological evidence in the nicoletiid genus Texoreddellia Wygodzinsky, 1973 (Hexapoda: Zygentoma: Nicoletiidae) in Texas and Mexico. Pp. 87–110 in Colkendolpher, J.C., & Reddell, J.R., eds. Studies on the Cave and Endogean Fauna of North America, Volume V.