Subterranean Biology 15: |—10 (2015)

doi: 10.3897/subtbiol. 15.8468 RESEARCH ARTICLE

http://subtbiol.pensoft.net

| Published by
The International Society
for Subterranean Biology

Cave dwelling Onychophora from
a Lava Tube in the Galapagos

Luis Espinasa', Radha Garvey’, Jordi Espinasa’, Christina A. Fratto’,
Steven J. Taylor’, Theofilos Toulkeridis®, Aaron Addison’

I School of Science, Marist College. Poughkeepsie, New York, USA 2 Arlington High School. Lagrangeville,
New York, USA 3.New Paltz High School. New Paltz, New York, USA 4 Sacred Heart University. Fairfield,
Connecticut, USA § Illinois Natural History Survey, University of Illinois, 1816 S Oak St, Champaign, IL
USA 6 Universidad de las Fuerzas Armadas ESPE, Campus Sangolqut. Sangolqut, Ecuador 1 Washington
University in St. Louis. St. Louis, Missouri. USA

Corresponding author: Luis Espinasa (luis.espinas|@marist.edu)

Academic editor: O. Moldovan | Received 23 August 2014 | Accepted 3 December 2014 | Published 21 January 2015
Attp://zoobank.org/35D306C9-76D6-4CAC-868D-C688331273FA

Citation: Espinasa L, Garvey R, Espinasa J, Fratto CA, Taylor SJ, Toulkeridis T, Addison A (2015) Cave dwelling
Onychophora from a Lava Tube in the Galapagos. Subterranean Biology 15: 1-10. doi: 10.3897/subtbiol. 15.8468

Abstract

A new population of velvet worms (Onychophora) inhabiting a lava tube cave in the island of Santa
Cruz, Galapagos, is reported here. The population size is large, suggesting that they may be troglophilic.
Its members are darkly pigmented, with no obvious troglomorphic features. Their 16S rRNA sequence
showed no differences when compared to an unidentified species of surface velvet worm from the same
island, thus supporting cave and surface populations belong to the same species. Based on the 16S rRNA
data, the Galapagos velvet worms derived from an Ecuadorian/Colombian clade, as would be expected of
ease of dispersal from the nearest mainland to the Galapagos Islands.

Keywords
Onychophora, Velvet worms, Galapagos, Santa Cruz, Lava tube, Troglophile, Troglobite, 16S rRNA

Copyright Luis Espinasa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

2 Luis Espinasa et al. / Subterranean Biology 15: I-10 (2015)

Introduction

The Onychophora, or velvet worms, are considered “living fossils” and among the most
interesting groups of animals for evolutionary biologists. Fossils dating back to 540 million
years show an overall anatomy which has remained largely unchanged. Extant taxa resem-
ble early Cambrian lobopodians, such as Aysheaia pedunculata (Walcott, 1911) from the
Burgess Shale formation in Canada (Whittington 1978) and Onychodictyon ferox (Hou,
Ramskold and Bergstrém, 1991) from the Chengjiang fauna of China (Ou et al. 2012).
The phylum Onychophora unites the primitive features of “worms,” such as Nemotoda
and Nematomorpha, with those of the arthropods (Grimaldi and Engel 2005), helping to
reconstruct the ancestral arthropod. Modern velvet worms are classified into two families,
Peripatidae and Peripatopsidae. ‘The early diversification of these groups pre-dates the
break-up of Pangaea, maintaining regionalization even in landmasses that have remained
contiguous throughout the history of the groups (Murienne et al. 2014). The species of
the Peripatopsidae are typically found in southern latitudes and show a classic Gondwa-
nan distribution; Australia, New Guinea, Chile, South Aftica, and New Zealand. The
Peripatidae have a more equatorial distribution and are restricted to the Neotropics, the
Antilles, West Africa, and areas of South-East Asia (Oliveira et al. 2013).

Two velvet worm species, the first a Peripatopsid, Peripatopsis capensis (Grube,
1866), and the second a Peripatid, not identified to species but labeled as Oroperipatus
sp. (Herrera 2014), have been reported in the Galapagos Islands. For the first species
there is no data regarding from which specific island where they were found, and the
second was collected in Santa Cruz (Herrera 2014). The Galapagos Islands are located
in the eastern Pacific Ocean, about 1,000 km west of the Ecuadorian mainland (Fig.
1A). These Islands were formed by shield volcanoes whose immense piles of basal-
tic lava flows have built up from the sea floor. The Galapagos hot spot represents a
process of magma supply which has existed for more than 90 million years while the
lithospheric plate has moved many thousands of kilometers in the same time interval,
carrying the hot spot-generated volcanoes away (Hoernle et al. 2002; Werner et al.
2003). Because these oceanic islands have remained unconnected to the continental
landmasses, the velvet worms must have dispersed to the Galapagos at some point, for
example, by rafting transport or accidentally by humans. The oldest island currently in
the Galapagos Archipelago is South Plaza, which has an estimated age of 4.2 million
years (+/- 1.8). However, the archipelago had islands at least 8 million years old that
have since eroded, sunk and whose remains are now seamounts on the Carnegie Ridge
(Christie et al. 1992). The island of Santa Cruz, where the velvet worms used for this
study were collected, has an estimated age between 0.7 and 1.5 million years.

In the volcanic islands of the Galapagos there are numerous lava tubes. Lava tubes
form when a top layer of flowing lava cools while the molten lava beneath the surface
continues to flow. As the flow subsides, the lava will empty out forming a lava tube.
While a variety of organisms inhabit Galapagos lava tube caves, relatively few are con-
sidered to be troglobitic (Peck and Finston 1993). Troglobites, or cave adapted organ-
isms, often lack eyes, cuticular pigmentation, have elongated appendages, and occur

Cave dwelling Onychophora from a Lava Tube in the Galapagos 3

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Figure |. A Location of the Galapagos Islands B Archipelago of the Galapagos. Samples collected were
from the island of Santa Cruz C Yellow pins indicate the surface locality and Kubler cave where samples
were collected D In blue, overlaid contour of the map of Kiibler cave. Notice that the cave is within the
city limits of Puerto Ayora.

in small, isolated geographic ranges. Troglobites most often evolved and derived from
non-troglobitic ancestors living in dark and humid habitats (Holsinger 1988).

The velvet worms are terrestrial and prefer dark environments with high air hu-
midity. They are found particularly in the rainforests of the tropics and temperate
zones where they live among moss cushions, leaf litter, under tree trunks and stones, in
rotting wood, in termite tunnels or in crevices in the soil into which they can withdraw
during the day (Grimaldi and Engel 2005). Their ability to squeeze themselves into
the smallest cracks and propensity to live in dark habitats would support the idea that
velvet worms are exaptated to colonize the cave environment. Additionally, they may
be evolutionarily co-opted to become successful troglobites. However, only two troglo-
bitic Onychophora are known: Peripatopsis alba Lawrence, 1931 (Peripatopsidae) of
South Africa and Speleoperipatus spelaeus Peck, 1975 (Peripatidae) from Jamaica.

During road work on the main highway near Puerto Ayora, the main town on the
Galapagos island of Santa Cruz, the roof of a lava tube collapsed uncovering a large cave.
During mapping and exploration of the cave, several velvet worms were discovered. The
purpose of this paper is to document this unique population and establish how this cave
population is related to other surface Onycophora through 16S rRNA sequencing.

4 Luis Espinasa et al. / Subterranean Biology 15: I-10 (2015)

Methods

This study is part of a larger project focused on cave mapping, geological investiga-
tions and cataloging biological resources of lava tube caves in the Galapagos (Taylor
et al. 2012). Within the cave, specimens were found crawling on the floor of the cave.
Surface specimens were found under a rock of a forested portion of Santa Cruz island.
Six cave specimens from Cueva de Kitibler (Near El Mirador de los Tuneles, within
the city limits of Puerto Ayora [Fig. 1C—D], Santa Cruz, Galapagos. $00°43'55.57",
W90°19'41.2074", mas] 18/03/2014. L. Espinasa, G. McDaid, R. Toomey and G.
Hoese cols.) and two from a surface locality near Rancho Primicias (El Chato, Santa
Cruz, Galapagos. S00°40'21.96", W90°25'52.20", 240 masl. 19/03/2014. L. Espinasa
and R. Espinasa cols.) were collected by hand and deposited in 100% ethanol.
Collecting and exportation permit #094-2014DPNG for project PC-64-14 to
Dr. Theofilos Toulkerdis was provided by Direccién del Parque Nacional Galapa-
gos and Ministerio del Ambiente, Ecuador. Genomic DNA samples were obtained
following standard methods for DNA purification using Qiagen’s DNeasy® Tissue
Kit, by digesting one lobopod leg of the individual in the lysis buffer. Markers were
amplified and sequenced as a single fragment using the 16Sar and 16Sb primer pair
for 16S rRNA (Edgecombe et al. 2002). Amplification was carried out in a 50 ul
volume reaction, with QIAGEN Multiplex PCR Kit. The PCR program consisted
of an initial denaturing step at 94 °C for 60 sec, 35 amplification cycles (94 °C for
15 sec, 49 °C for 15 sec, 72 °C for 15 sec), and a final step at 72 °C for 6 min ina
GeneAmp® PCR System 9700 (Perkin Elmer). PCR amplified samples were puri-
fied with the QIAquick PCR purification kit and directly sequenced by SeqWright
Genomic Services. Chromatograms obtained from the automated sequencer were
read and contigs made using the sequence editing software Sequencher'™ 3.0. All
external primers were excluded from the analyses. BLAST was used to identify Gen-
Bank sequences that resemble the Galapagos specimens. Sequences were aligned

with ClustalW2.

Results

Kiibler Cave (El Mirador) has a horizontal length of 914 m and a depth of 70 m. It ex-
tends beneath land currently being developed for human habitation at the outer edge
of Puerto Ayora (Fig. 1D). Velvet worms found inside the cave were darkly pigmented,
had eyes, and showed no apparent troglomorphic features (Fig. 2). Nonetheless, they
do not seem to be accidentals as the cave appears to host a large population of velvet
worms. [welve specimens were observed in a single trip. Specimens ranged in size from
0.7 cm to 3.0 cm, suggesting a breeding population is present. Individuals appeared to
be in good condition, moving swiftly across the terrain. One was seen apparently feed-
ing on a terrestrial isopod (Oniscoidea) (Fig. 3). Live specimens, their behavior, and
the Kiibler cave environment can be seen at https://www.youtube.com/watch?edit=vd

Cave dwelling Onychophora from a Lava Tube in the Galapagos 5

Figure 2. A typically dark-pigmented individual from the population of velvet worms inhabiting Kubler

Cave on Santa Cruz island, Galapagos Islands.

&v=M9ZTcRLjknw. Live specimens from the El Chato surface locality (Fig. 4) can be
seen at https://www.youtube.com/watch?v=8EXaZwcBGuw®&edit=vd.

When the DNA of the six cave specimens and two surface specimens was se-
quenced, their 16S rRNA was found to be identical (GenBank # KM102162 and
KM102163). The fragment sequence was 455 bp long. When a BLAST analysis was
performed, Epiperipatus sp. (KC754524) from Colombia appeared to be the most
similar, differing by 62 bp (13.6%), followed by two specimens of an unidentified ge-
nus of Peripatidae (KC754526 and KC754520) from mainland Ecuador, from which
they differed by 68 bp (14.9%) and 77 bp (16.9%) respectively. Our sequences were
also compared against velvet worm species akin to that previously reported for the
Galapagos Islands. Two specimens not identified to species but labeled as Oroperipatus
sp. (NC015890.1 and JF800076.1) have been sequenced for their 16S rRNA. The
Galapagos specimens differed from them by 83 bp (18.2%). Finally, when compared
against Peripatopsis capensis (KC754566), they differed by 113 bp (24.8%).

Discussion

A population of velvet worms (Onychophora) has been found in a lava tube cave on
the island of Santa Cruz, in the Galapagos Archipelago. The American Peripatids are
roughly divided into two groups which diverged over 145 million years ago in the
Mid-Jurassic; a clade with Ecuadorian and Colombian species and a clade centered
mainly in and around the Caribbean (Murienne et al. 2014). When analyzing to which

6 Luis Espinasa et al. / Subterranean Biology 15: I-10 (2015)

*

Figure 3. Within the lava tube food web, cave velvet worms appear to be successful predators. This cave

.

specimen was observed apparently feeding on a pill bug (Isopoda: Oniscoidea: Armadillidae). The cave
population of the onlychophoran is thought to be large, estimated in at least the hundreds. (Photo by
Rickard S. Toomey II).

subgroup the Galapagos velvet worms are most closely related, based on our 16S rRNA
data, they were included within Ecuadorian/Colombian clade of Peripatids.

The cave specimens’ 16S rRNA are identical to that of surface velvet worms col-
lected from the same island, strongly suggesting that both cave and surface populations

Cave dwelling Onychophora from a Lava Tube in the Galapagos oF

Figure 4. A velvet worm from beneath a stone on the surface on Santa Cruz island, Galapagos Islands.
The 16S rRNA of these animals was found to be identical to those found in Kiibler Cave.

belong to the same species. Despite cave specimens being darkly pigmented and having
no obvious troglomorphic features, the observation of twelve individuals comprising
both juveniles and adults, indicated that the population in Kiibler Cave (El Mirador)
is large. Only a small proportion of the cave could be scanned, especially because in
volcanic terrains there are innumerable crevices and rocks where individuals could
hide. It is likely that the population is at least in the hundreds, but this is only a rough
estimate. Furthermore, specimens appeared to be as healthy and mobile as their surface
counterparts. Velvet worms, with their propensity to live in dark habitats, are exap-
tated to colonize the lava cave habitat and evolutionarily co-opted to become successful
troglophiles and troglobites, as reported by Clarke (2010) who has described velvet
worms inhabiting lava tubes in the Undara system in Australia. We therefore propose
assigning these Galapagos velvet worms a status of “troglophile”.

While we have determined that both cave and surface velvet worms collected for
this study most likely belong to the same species, some questions still remain: To which
species does the Santa Cruz island Onychophora belong? ‘There are two velvet worms re-
ported as occurring on the Galapagos Islands, Oroperipatus sp. and Peripatopsis capensis.
The latter species, the Cape velvet worm, is a habitat specialist peripatopsid character-
ized as having a low dispersal capability and sensitivity to dehydration, restricted to the
Cape Peninsula in South Africa (McDonald and Daniels 2012). Its presence within the
Galapagos Islands is most likely accidental and human facilitated. The specimens sam-
pled in this study do not belong to this species as evidenced by the substantial sequence

8 Luis Espinasa et al. / Subterranean Biology 15: I-10 (2015)

difference (113 bp; 24.8%) between their 16S rRNA. Sequence showed them instead to
belong within the peripatids.

Our Galapagos specimens also showed significant difference (83 bp; 18.2%) from
the specimens available in GenBank of genus Ovoperipatus. Instead, they showed the
least difference (62 bp; 13.6%) with a member of genus Epiperipatus from Colombia and
with specimens of an unidentified genus of Peripatidae from Ecuador (68 bp; 14.9%).
This information could be interpreted as our specimens belonging to a different genus
from the velvet worms previously reported for the Galapagos. However, cladistic analysis
of the Onychophora shows that the American genera are oftentimes paraphyletic (Muri-
enne et al. 2014), and that taxonomic species do not group by their genera in the phyletic
tree obtained with the 16S rRNA. Thus, our unidentified specimens could still belong to
the same genus/species as those previously reported for the Galapagos Islands.

Another possibility is that Herrera’s (2014) identification of some Galapagos vel-
vet worms as belonging to Oroperipatus sp. could be incorrect. Low character variation
among Onychophoran species has been an obstacle for taxonomic and phylogenetic
studies (Oliveira et al. 2012). Almost 200 species of Onychophora have been described.
However, the validity of many of these taxa is uncertain and ~10% of the described
species of Onychophora show major taxonomical problems and should be regarded as
nomina dubia (Mayer and Oliveira 2011). Future studies based on morphology and
sequence data may resolve the identity of the cave-inhabiting Oncychophora of Kiibler
Cave, allowing assignment to a previously described species of Oroperipatus or Epiperi-
patus, or, possibly, to an undescribed species.

Acknowledgments

Partial support for the project came from two National Speleological Society grants;
the Education and the Conservation grants. The Subterranean Ecology Institute pro-
vided additional support for fieldwork. Travel expenses for LE were supported by a
VPAA grant from Marist College. Further financial and logistic support was given by
ESPE project # 2014-PIT-012. Research and exportation of samples permit was given
by the Galapagos National Park (PC-64-14 and 094-2014 DPNG). G. McDaid, R.
Toomey, G. Hoese and B. Osburn helped exploring, mapping and/or collecting the

specimens. We also thank the two anonymous reviewers for their suggestions.

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