The cave-dwelling dipluran ( Diplura , Campodeidae ) on the edge of the Last Glacial Maximum in Vancouver Island caves , North America ( Canada )

A new cave-dwelling dipluran of the North American endemic genus Haplocampa is described, coming from a couple of caves excavated in a small limestone karstic area near Port Alberni, Vancouver Island (British Columbia, Canada). To Haplocampa belong five soil-dwelling species. L. M. Ferguson cited no less than eight more species living in soil and cave habitats in several US states but without producing any formal descriptions. Haplocampa, in spite of its large lateral crests on the unequal claws, has clear taxonomical features as a Campodeinae and is closely related with the cave-dwelling Pacificampa and Eumesocampa genera, due to sharing similar macrosetae body distribution and absence or reduction of the lateral process. The new proposed species, Haplocampa wagnelli Sendra, sp. n., is rather interesting for its troglomorphic features: antennae with 32 antennomeres; olfactory chemoreceptors, each a multiperforated, folded-spiral structure; and numerous gouge sensilla. In addition, it is one of the northernmost troglomorphic species to have colonised – presumably recently – an area occupied by the Late Wisconsinian North America ice sheet during the Last Glacial Maximum. Furthermore, the close affinities between Haplocampa, Pacificampa (from caves in the extreme east of continental Asia and the southern Japanese Islands), Metriocampa (from the east of Asia and North America) and Eumesocampa (endemic to North America) suggest probable dispersal events over the Bering Land Bridge. Subterranean Biology 29: 59–77 (2019) doi: 10.3897/subtbiol.29.31467 http://subtbiol.pensoft.net Copyright Alberto Sendra, Craig Wagnell. 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. RESEARCH ARTICLE Subterranean Biology Published by The International Society for Subterranean Biology A peer-reviewed open-access journal


Introduction
Although subterranean diplurans were already known from North America since the 19th century (Packard 1871), few species have been described (Condé 1949;Condé and Bareth 1996;Ferguson 1996;Sendra et al. 2016;Wygodzinsky 1944) and many remain undescribed despite a vast sampling effort.Haplocampa genus is a clear example.Haplocampa is endemic to North America (Silvestri 1911); it was first discovered in Shasta Springs, California, with its type species Haplocampa wheeleri Silvestri, 1911.Later, the same entomologist (Silvestri 1933) described three new species: Haplocampa rugglesi Silvestri, 1933;Haplocampa chapmani Silvestri, 1933;and Haplocampa drakei Silvestri, 1933.One more species of this genus could be included if we consider the form Haplocampa cf.chapmani by Condé and Geeraert (1962).These five species live in soil habitats and are distributed in Montana, Oregon and Washington in the USA and Alberta in Canada.During the 1980s and 1990s, a large sampling effort was carried out in 281 caves in California alone (Elliott et al. 2017) revealing 3 or more species of Haplocampa, andFerguson (1981, 1992) reported on 7 or 8 species of Haplocampa from 21 lava tubes mainly in the Pacific Northwest.Ferguson (2009) also reported on his identification of 9 new species of Haplocampa from the caves of Colorado, bringing the total number of identified species of Haplocampa from caves to 26.Nevertheless, no formal description was made until now and many Haplocampa specimens remain undescribed in American museums and invertebrate collections (Graening et al. 2014).In 1996, the Central Island Caving Club started wandering the steep forested slopes of Vancouver Island.Since that time, the Club has mapped over 20 new caves, including what is now the most popular recreational cave on Vancouver Island.Thanks to the Club's great work, the explorations of a group of cavers from Vancouver Island (British Columbia, Canada) have given us the opportunity to describe the next new species of Haplocampa after more than half a century.

Sampling methods
Fauna collections were carried out in two sites of Fossli Slot #2 and one site from Kiku Pot (see below the description of caves).Sampling was focused on regions where visible signs of chewed-up organic matter could be seen.Collections were made manually using #00 brushes and kept in small vials with 90% ethanol.Once the specimens were spotted, they were dabbed with a brush moistened with ethanol and immediately placed into vials, labelled and sealed for transport.

Material processing and identification
The specimens were washed using distilled water and were put between slides and glass coverslips to be examined under a phase-contrast optical microscope (Leica DMLS) using Marc André II solution.The illustrations were made with a drawing tube and the measurements were taken with an ocular micrometre.For measuring the body length, the specimens were mounted 'in toto' and were measured from the base of the frontal process distal macrochaetae to the abdomen's supra-anal valve.Two paratypes were coated with palladium-gold used for scanning electronic microscopic photography (Hitachi S-4100) and measurement of the sensilla.
The morphological descriptions and abbreviations used in this article follow Condé (1956).We use gouge sensilla for the concavo-convexly shaped sensilla located on the antennae and described by Bareth and Condé (1981), the function of which is still unknown, and rosette gland formations for the epicuticular glands described in several Campodeinae species (Bareth and Juberthie-Jupeau 1996).Description.Body length 4.4 mm (male) and 3.4-6.0mm (females).Epicuticle smooth under optical microscope but reticulated in high magnifications in round polygonal structures variable in size (Figs 4,(14)(15); rosette gland formations present along the body (Figs 5,16); body with abundant short and smooth clothing setae.

Haplocampa wagnelli
Moliniform antennae.Every intact antenna in the six type specimens has 32 antennomeres; from 0.5 to 0.7 times longer than body in larger and smaller adults, respectively.First antennomere four times shorter than second antennomere, and apical antennomere 1.3 longer than wide (Fig. 1); the other antennomeres, as long as wide (Fig. 7).Cupuliform organ occupying 1/3 of the total length of the apical antennomere with about five complex olfactory chemoreceptors, each one a multiperforated-folded spiral structure, all tightly packed in the narrow open space of the cupuliform organ (Figs 2, 3).Distal and central antennomeres with a sensorial equipment: one whorl of bifurcated macrosetae, two to three whorls of untidy setae , and a single distal whorl of 10-13 short and thick gouge sensilla of 14-16 µm long 2-3 very short grooved closed-bud form sensilla 6-8 µm long; this sensorial equipment is also present in the apical antennomere (Figs 4,(6)(7)(8)(9)(10).Proximal antennomeres with typical trichobothria plus a thick and long sensillum on the third antennomere, located in ventral position, although the ventral c and d macrosetae are not differentiated.
Head subtrapezoidal with slightly protuberant lateral posterior angles (Fig. 11).Plain frontal process with the slightly differenced frontal smooth macrosetae (Fig. 12).From the three macrosetae along each side of the line of insertion of antennomere and x setae, a macrosetae is not differentiated and i/p/x have 36/57/23 relative lengths, all smooth (Fig. 12).Suboval labial palps with latero-external long thick sensillum, with two guard setae, up to 8 setae on anterior border and up to 80 neuroglandular setae.
Thoracic macrosetae distribution (Fig. 13): pronotum with 1+1 ma, 1+1 la, 1+1 lp; mesonotum with 1+1 ma, 1+1 la, 2+2 lp 2, 3 ; metanotum with 1+1 ma, 1+1 lp 2 macrosetae.All macrosetae relatively well developed, long with thin long barbs; marginal setae slightly longer and thicker than clothing setae and also smooth.Legs very slightly elon-gated, metathoracic legs reaching the VIII abdominal segment in smaller adults and the VI in larger ones.Femur and tibia similar in length but tarsus clearly shorter and thicker (tibia/tarsus ratio 0.6 to 0.7) (Table 1).Femur I-III with one dorsal macrosetae well differentiated with a few thin long barbs in its distal half.Calcars with a few thin, long barbs throughout.Tibia I-III with two or three ventral macrosetae bifurcated in the apex (Fig. 17).Three or two dorsal, lateral and sometimes ventral tarsal setae similar to clothing setae but much longer (Fig. 18).Unequal claws (posterior claw 1.5 longer than anterior) with large lateral crests and no lateral process; ventral side of the claws noticeably ridged and covered by a micro-granulation surface; a spiny protrusion is visible in the basal portion of both claws (Figs 18-21); posterior claw with a large backward overhang (Figs 18,19).
Urosternite I with 6+6 macrosetae ; II to VII with 4+4 macrosetae; VIII with 1+1 macrosetae; short to middle size urosternal macrosetae, bi or trifurcated or with long barbs.Short styli with smooth short apical setae with two long teeth;  bifurcated subapical and ventromedial setae (Fig. 25).Cerci half shorter than the body length, from 0.64× in the smaller adults to 0.47× in the holotype; so, the length of the cerci increases disproportionally with the body size.As well, the length of the articles increases from proximal to distal.Basal article is divided into three to eight secondary articles, followed by five to six primary articles bearing from two to five whorls of     bifurcated macrosetae, and three to six smooth setae including the distal setae whorl in each primary article (Table 2; Figs 26, 27).Female urosternite I with short subcylindrical appendages, each bearing up to 30 a 1 -glandular setae in a distal field (Fig. 23).
Male urosternite I with short moderately thick subcylindrical appendages, each bearing about 50 a 1 -glandular setae in a large field; up to setae covered the with two to three rows the posterior part of the first urosternite (Fig. 24).
Remarks.The most visible features are the slightly troglomorphic characteristics of H. wagnelli sp.n., as shown by its antennae with 32 antennomeres, a record within the genus Haplocampa but a moderate antennomere number in a troglomorphic campodeid; and the five complex olfactory chemoreceptors, each one a multiperforated, folded-spiral structure However, any comparison with other Haplocampa species is currently impossible since these sensilla have not been described in any other species of the genus.The closest species to H. wagnelli sp.n. is H. rugglesi from Mount Rainier (Washington, USA), with equal macrosetae distribution on nota and similar on urotergites.Nevertheless, some taxonomic features are unique to H. wagnelli sp.n., such as medial anterior macrosetae on urotergites and three tibial ventral macrosetae.Many taxonomical details remain incomparable what is known described species, due to the lack of high magnifications.

Description of type localities (caves)
The caves are all located near the town of Port Alberni, Vancouver Island, British Columbia, Canada .The limestone area covers roughly 2400 hectares, and within that area lay over 20 hidden caves, as well as many resurgences, dry sinks, small pits and fissures.Only a short drive and an easy hike are required to reach most caves, providing great opportunities to turn this area into an outstanding caving recreational site if properly managed.

Fossli slot caves
Fossli #1 (L63.5 D25): swallet entrance with active surface stream.First sightings of H. wagnelli sp.n. on 5 th April 2005.Fossli #2 (L67.5 D26.5): small slot entrance filled with rotten wood.Upper samples collected 15 th July 2018, 30 m from the entrance in a moist bed of sandy organic debris.Lower samples collected 6 th August 2018, 60 m from the entrance in a moist bed of sandy organic debris 2 m above sump.H. wagnelli sp.n. mud workings visible at both sites.
Kiku Pot cave, (L1489 D92): Breakdown entrance with active stream passage throughout system.H. wagnelli sp.n. first sightings August 2017 while surveying.Collection taken 5 th August 2018, at bottom waterfall just below where water disappears approximately 150 m from the entrance on a medium.A medium sized bed of sandy organic material with scattered woody debris.H. wagnelli sp.n. workings were visible over 75%.Area floods during high water.Many mud workings visible on muddy shelves higher in passage.At the time of collection, an adult fungus gnat was noticed hopping around.

Cave Convervation Vancouver Island
Vancouver Island is known to have over 1600 caves, with more mapped and explored caves than the rest of Canada combined.Some caves reach over 10 km, and some host unique geological, palaeontological, archaeological and biological features.Vancouver Island caves are mostly active caves, with streams and rivers running through them most of the year.The caves help the streams to maintain constant water temperatures year round and a proper pH, which increases water quality for fish and wildlife.So far, little has been done to protect the caves from poor logging, mining and recreational practices.All caves west of the Port Alberni area are easily accessed by the public and none so far have protection or proper management.Unfortunately, some already have seen misuse.More needs to be done in the future if we want to protect this special resource.

Phylogenetic affinities of the genus Haplocampa with notable biospeleological and biogeographical comments
In Haplocampa species, their unequal claws have large lateral crests, as do many genera of Plusiocampinae.Applying the right criteria, Paclt (1957) included Haplocampa within Campodeinae.In fact, many taxonomical features of Haplocampa are held by Campodeinae, such as the distribution and number of pronotum macrosetae, with at most 3+3 medial anterior, lateral anterior and lateral posterior macrosetae.Other notable features of Haplocampa are shared with many Campodeinae genera, namely, epicuticle with rosette gland formations; plain frontal process; number and distribution of urotergal macrosetae with medial macrosetae and a maximum of three pairs of lateral posterior macrosetae; simple stylus setae; and number and distribution of urosternal macrosetae, with up to eight pairs of macrosetae on first the urosternite, up to five pairs of macrosetae on the second to seventh urosternites and one pair on the eighth.
In all likelihood, Pacificampa Chevrizov, 1978 is the most closely related to Haplocampa, given the strong similarities in the number and distribution of the macrosetae body and the absence of lateral pretarsus processes, with one important difference: the lack of lateral crests in Pacificampa (Chevrizov 1978;Sendra et al. 2018).The same differential feature is shown by other related genera of Haplocampa, in Metriocampa Silvestri, 1912 andEumesocampa Silvestri, 1933.All these four genera can be found in the East of Asia and in North America, on both sides of the northern Pacific Ocean, which suggests dispersal events over the Bering Land Bridge.This hypothetical palaeobiogeographical distribution was suggested by Ferguson (1997) for Eumesocampa, Haplocampa, Pacificampa and Plutocampa Chevrizov, 1978.Another perhaps more parsimonious hypothesis could be that they had an ancient Laurasian distribution and their current distribution reflects a vicariance event after the breacking of the Bering Bridge (Loris Galli pers.suggestion).
Haplocampa and Pacificampa share ecological similarities, and phylogenetic, too.Both are present in subterranean ecosystems.In the case of Pacificampa, all of the five known species have been found in caves and can be considered troglobites.Three of these species can be found in the north of the Korean Peninsula (Chevrizov 1978;Ferguson 1997) and two were recently described in two southern Japanese Islands (Sendra et al. 2018).In Haplocampa, the five described species have been found within the soil at high-altitude localities.However, Ferguson (1981) suggested no less than eight undescribed species of Haplocampa from caves located in the Ozarks karstic region at the centre of North America and in the small volcanic and karstic areas of the Pacific coast.Ferguson (1981) remarked that in the case of Haplocampa, Eumesocampa and Tricampa Silvestri, 1933, 'All three have epigean members at high latitudes or high altitudes in the western mountains, and cavernicolous members at lower latitudes and altitudes'.Nevertheless, Haplocampa wagnelli sp.n. is an exception to Ferguson's comment (1981).
H. wagnelli sp.n. is a slight troglomorphic species and several features show this.Among these features are the very slight elongation of antennae, 0.5× to 0.7× the body length, with moniliform antennomeres, and the cerci 0.5× to 0.6× the body length.In troglomorphic campodeids, the length of antennae can double that of the body, with always more than 30 antennomeres, and the cerci can be two or three times the body length; there is also an increase in the number of the cercal articles.Furthermore, troglobitic campodeids have elongated legs, reaching the end of the abdomen and a slim body.All of these features are trademarks of highly adapted subterranean campodeid species (Sendra et al. 2017).However, in H. wagnelli sp.n., its legs reach the VI to VIII abdominal segment and the body is not slender.But the trogloniont condition of H. wagnelli is supported by the presence of 32 antennomeres, five complex, multiperforated, folded-spiral sensilla, and a high number (14-16) of thick gouge sensilla.These are solid evidences for subterranean adaptations.For these reasons, H. wagnelli sp.n. can be considered to be a slightly troglobiont species adapted to live in caves and other subterranean spaces, but it could probably also be found in soil habitats.This is the case for the soil-dwelling species H. drakei, with 26 antennomeres, found nearby in Banff National Park, Alberta, Canada (Silvestri 1933).
H. wagnelli sp.n. may represent one of the most northerly cave-dwelling adapted dipluran species being found at 49° latitude north.The most northerly cavedwelling species is Litocampa hubarti Bareth, 1999, found in Grotte Lyell (Liège, Belgium) at 50° latitude north, and like H. wagnelli sp.n., it shows humble morphological subterranean-adapted features (Bareth 1999).Both species are geographically in the limits of the Last Glacial Maximum.Furthermore, in the case of H. wagnelli sp.n., its current distributional area was under the Cordilleran Ice Sheet the Late Wisconsinian North American ice sheet complex, during the Last Glacial Maximum 18 ka BP (Dyke 2004).Several hundred years later the retreat of the glacial ice occurred, and as a consequence, the subterranean habitat colonisation of Vancouver Island was possible.

Figures 6 -
Figures 6-10.Haplocampa wagnelli Sendra, sp.n. 6 distal gouge sensilla whorl in a medial antennomere 7 medial antennomere 8 detail of the ending portion of a gouge sensillum 9 detail of external side of a gouge sensillum 10 detail of lateral side of a gouge sensillum.

Figures 29 -
Figures 29-33.29 Map of Fossli Slots caves with spots where Haplocampa wagnelli sp.n. was seen 30 entrance of Fossli Slot #2 cave 31 Kiku Pot cave with spots where Haplocampa wagnelli sp.n. was seen 32 entrance of Kiku Pot cave with Felix Ossigi-Bonanno and Craig Wagnell after the success finding 33 entrance of Kiku Pot cave viewed from inside the cave.

Sendra, sp. n.
Etymology.This species is dedicated to the co-author of this article, a caver who has dedicated many years sampling and exploring in Vancouver Island caves.

Table 1 .
Haplocampa wagnelli Sendra, sp.n., length of the body, antennae and metathoracic leg including their segments, the cerci (units in mm), and the number of antennomeres.

Table 2 .
Haplocampa wagnelli Sendra, sp.n., length of cercal articles and total length (units in mm) including number of articles of each cercus.