New oversize troglobitic species of Campodeidae in Japan (Diplura)

Two new oversize troglobitic species of diplurans campodeids, Pacificampa daidarabotchi Sendra, sp. n. and Pacificampa nipponica Sendra, sp. n., found in three caves in two southern Japanese islands are described. It is the first record of cave-dwelling Diplurans from Japan and more specifically these are two Campodeinae of Pacificampa Chevrizov, 1978 formally known in continental Asia with three cave-dwelling species described in Russia and China near the north of the Korean peninsula. In P. daidarabotchi sp. n., in addition to its oversized body, the longest ever known in campodeids family, it shows as its main differential taxonomical feature the absence of lp metanotal macrosetae. P. nipponica sp. n. is much closer to continental Pacificampa species but it shows differences in the urotergal macrosetae formula. Both Japanese species have clear troglomorphic features related with their elongated body and appendages and an increase in number and complexity of the sensorial antennal equipment with unique olfactory chemoreceptors. Two biogeographical remarks are inferred: P. nipponica sp. n., has been found in two islands that were connected during the glacial age and P. daidarabotchi sp. n. lives in a single cave near another where P. nipponica sp. n. dwells which allowing to suggest a sympatric distribution; Pacificampa seems to be related with Eumesocampa, a genus from North-America, as a consequence of the Holarctic connection between Asia and America. The interests of P. daidarabotchi sp. n. and P. nipponica sp. n. as endemic species in addition to their biogeographical importance should be taken into consideration to ensure better management of the three touristic caves where they dwell and especially the protection of Mejido-do cave, the only location of the large P. daidarabotchi sp. n. near to an active quarry. Subterranean Biology 27: 53–73 (2018) doi: 10.3897/subtbiol.27.28575 http://subtbiol.pensoft.net Copyright Alberto Sendra 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. RESEARCH ARTICLE Subterranean Biology Published by The International Society for Subterranean Biology A peer-reviewed open-access journal


Introduction
To date no data is available about subterranean dipluran fauna in Japanese caves or any subterranean habitats.The knowledge of diplurans in Japan is limited to soil habitats, the first one was a japygid described by Enderlein (1907).About twenty years later Kuwayama (1928) and Silvestri (1928) added seven species for Japanese islands including two Parajapygidae ones.In 1931 Silvestri described five species of Campodeidae, four within Campodeinae and one within Lepidocampinae, most endemic to Japan.
No species of Diplura have described from that moment on, until when an expedition to caves in Japan was carried out from September 5 to 15, 2017.The main objective of this expedition was to sample cave invertebrates in order to understand how communities respond to habitat traits (manuscript in prep.).Eleven carbonate caves were sampled (three on Shikoku Island and eight on Kyushu Island).In Shikoku island, the following caves were sampled: Hiura-do cave (Kumakogen municipality), Inaba-do cave (Tsuno municipality) and Rakan Ana cave (Seiyo municipality).In Kyushu island, the following caves were sampled: Goya Daini Shonyu-do, Goya Daiichi Shonyu-do, Merijo-do and Goya Daysan-do caves (all located in Kitakyushu municipality); Nichiko-do and Nichiko Ni-do caves (both located in Yatsushiro municipality) and Takazawa Shonyu do and Osê-do caves (both located in Kuma municipality) .

Sampling methods
The fauna collections were carried out both in transects and throughout several habitats of the caves, prioritizing areas (in the case of general collections) with accumulations of organic matter.The collections were done manually, using brushes and tweezers.The organisms were placed in 70% ethanol immediately after collection.Diplura specimens were found in only three caves (Mejiro-do, Goya Daiichi Shonyu-do and Inaba-do caves) in Kyushu and Shikoku Islands.

Material processing and identification
The specimens were washed using distilled water and were mounted on slides with Marc André II medium to be examined under a phase-contrast optical microscope (Leica DMLS).The illustrations were made with a drawing tube, and the measure-ments were taken with a micrometer ocular.To take measurements of body length, the specimens were mounted "in toto" and measured from the base of the frontal process distal macrochaetae to the abdomen's supra-anal valve.For scanning electron microscopy (Hitachi S-4100), two paratypes were coated with palladium-gold used for scanning electronic microscopic photography and measurement of the sensilla.
One intact antenna in the male paratype ME01 with 37 antennomeres, slightly (1.07) longer than the body (the other antennae of the same specimen is presumably regenerated with 28 antennomeres, 7.4 mm length) and another intact antenna in the young female ME03 with 41 antennomeres, 1.21 times longer than the body (Table 1).Apical antennae 4.4-4.7 longer than wide and central antennomeres 2.5 longer than wide Fig. 1).Cupuliform organ occupying 1/11 of the total length of the apical antennomere with 8-11 unique olfactory chemoreceptors each one with a complete caliciform pocket that encloses complex folds in radial expansions coming from a central axis, both of which are covered by small pores (Figs 2, 3).Distal and central antennomeres   with a sensorial equipment of macrosetae and setae in addition to a single distal whorl of 12-16 thin and long gouge sensilla (Figs 5,6) 42-34 µm long and a 2-3 very short coniform sensilla 8 µm long, also present in the apical antennomere (Fig. 4).Proximal antennomeres with typical trichobothria plus the sensillum of the third antennomere coniform and slightly thick located in ventral position between d and e macrosetae.
Plain frontal process with the three frontal macrosetae smooth.The three macrosetae along each side of the line of insertion of antennomere and x setae smooth and with 31/51/21/30 (a/i/p/x) relatives lengths.Suboval labial palps with latero-external short thick sensillum, with two guard setae, up to 12 setae on anterior border and up to 130 neuroglandular setae.
Thoracic macrosetae distribution (Fig. 7): pronotum with 1+1 ma, 1+1 la, 1+1 lp; mesonotum with 1+1 ma, 1+1 la, 2+2 lp 2, 3 (1+2 lp 3 -lp 2,3 in paratype ME01); metanotum with 1+1 sma, 1+0 ma in paratype ME01) macrosetae.All macrosetae relatively well developed but lp slightly thicker and longer than the others, and all with thin short barbs along the distal third or fourth; marginal setae longer thicker than clothing setae, smooth or with a few tiny distal barbs.Legs elongated, metathoracic legs reaching the IX abdominal in the adults (ME01 and ME02) and the end of the abdomen in the young female (paratype ME03) (Table 1).Femur I-III with one dorsal macroseta (Fig. 11) well differentiated but smooth.Calcars slightly differentiated with a few thin apical barbs (Fig. 12).The tibial two rows of ventral setae are longer and thicker than clothing setae but smooth like the clothing setae (Fig. 13).Tibia I-II with one ventral macroseta, tibia III with two ventral macrosetae (1+2 in paratypes ME01 and ME03) with a few tiny thin apical barbs.Dorsal and lateral tarsal setae similar to clothing setae but much longer (Fig. 15).Slightly unequal to subequal slightly curved claws (posterior claw 1.05-1.25 longer than anterior one); dorsal side of the claws have more pronounced ridges than the ventral side (Figs 14); any expansion of the claws is present.
Female urosternite I with subcylindrical appendages thinner than male appendages (2,7 times longer than wide), each bearing up to 55 a 1 -glandular setae in a distal field.
Male urosternite I (Fig. 18) with moderated thick subcylindrical appendages (2 times longer than wide), each bearing up to 90 a 1 -glandular setae in a large field covering almost a distal third of the appendage.
Spermatozoid fascicles present in the paratype male testis but difficult to observe.They are about 100 µm in diameter with a spiral round structure up to 4-7 µm in diameter with 3-5 turns.
Remarks.Although the former description of Pacificampa Chevrizov, 1978 in Russia near the north of the Korean peninsula had a poorly drawn text, the differential features of the genus were clear: simple subequal claws with no lateral telotarsal process, 3+3 (ma, la, lp) pronotum, 4+4 (ma, la, lp 2,3 ) on mesonotum and 2+2 (ma, lp 2 ) on metanotum with the exception of 1+1 ma in P. daidarabotchi sp.n.; one dorsal femoral macroseta and two ventral tibial macrosetae.Nevertheless, no description of the sexual features was cited in the diagnosis of the genus, nor in any of the proposed species (Pacificampa birsteini Chevrizov, 1978 andPacificampa caesa Chevrizov, 1978), perhaps this was due to their simplicity.In both sexes the first urosternal appendages are subcylindrical with distal a1 glandular setae slightly more abundant in males than in females.No other glandular setae are present in the first urosternite.
P. daidarabotchi sp.n. differs from other Pacificampa species in several features including its long body with 10.1 millimeters, that is the longest Campodeidae that has ever been recorded.An oversize body could be the rule in the rest of Pacificampa species although there is still not enough data to demonstrate this.Furthermore, it has longer antennae and cerci and more numerous antennomeres and cercal articles than other species of the genus; although the most notable features are the reduction of metanotal macrosetae with 1+1 ma (1+1 ma, 1+1 lp 3 in others species of the genus) and the reduction and variability in number of urotergal macrosetae.Description.Body length 5.0 and 6.3 mm (males), 6.0 mm (female) and 4.8 mm (juvenile) (Fig. 36-39, Table 3).Epicuticle smooth under optical microscope; body with abundant thin and smooth clothing.
One intact antenna in the male holotype SH01 with 34 antennomeres, longer (1.17) than the body.Apical antennae 4.6 times longer than wide and central antennomeres 3.5 times longer than wide (Table 3).Cupuliform organ occupying 1/10 of the total length of apical antennomere about 8 complex olfactory chemoreceptors.Distal and central antennomeres with a sensorial equipment of macrosetae and setae in addition to a single distal whorl of 12-14 thin and long gouge sensilla and 2-3 very short coniform sensilla, also present in the apical antennomere.Proximal antennomeres with typical trichobothria plus the sensillum of the third antennomere coniform located in dorsal position between b and c macrosetae.
Plain frontal process with the three frontal macrosetae smooth.The three macrosetae along each side of the line of insertion of antennomere and x setae smooth and with 25/40/26/20 (a/i/p/x) relative lengths.Suboval labial palps with latero-external short thick sensillum, with two guard setae, up to seven setae on anterior border and up to 110 neuroglandular setae.
Thoracic macrosetae distribution (Fig. 23): 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 3 macrosetae.All macrosetae developed slightly thick and with short barbs along the distal half or third; marginal setae longer and thicker than clothing setae with distal thin barbs.Legs elongated, metathoracic legs reaching the IX abdominal in the adults (ME01 and ME02) and the IX abdominal in the juvenile (paratype SH02) (Table 3).Femur I-III with one long dorsal macrosetae well differentiated with a few distal barbs.Tibia I-II with one ventral macroseta, tibia III with two ventral macrosetae with a few tiny thin apical barbs.Calcars differentiated with 1-3 long distal barbs.The tibial two rows of ventral setae are longer and thicker than clothing setae but smooth like the clothing setae.Dorsal and lateral tarsal setae similar to clothing setae but much longer (Table 3).Subequal slightly curved plain claws (posterior claw 1.05 longer than anterior one); no expansion of the claws is present.
Urosternite I with 6+6 macrosetae (5+5 in juvenile SH02); urosternites II to VII with 4+4 macrosetae; urosternite VIII with 1+1 macrosetae; almost all urosternal macrosetae long, well differentiated and covered by long barbs along the distal half.Smooth apical setae of styli with a short tooth with one tiny thin barb; also, smooth subapical and ventromedial setae, the ventromedial being much longer than the others.Cerci in the holotype with six articles including the basal article, slightly shorter than the body length (0.99); in juvenile paratype young SH02 has two cerci with six and seven articles, 1.30 and 1.41 longer than the body length; their basal articles show several whorls of long thin macrosetae with a few apical barbs, along the medial and distal articles these long macrosetae became smooth and whorls of sorther smooth setae are present   among them; all the articles are characterized by an apical whorl of thin smooth setae also present at the end of the last article (Table 4).Female urosternite I with subcylindrical appendages slightly thinner than male appendages 2.2 times longer than wide, each bearing 30 a 1 -glandular setae in a distal field.
Male urosternite I with moderated thick subcylindrical appendages 2 times longer than wide, each bearing up to 50 a 1 -glandular setae in a field covering the distal fifth of the appendage.Remarks.P. nipponica Sendra sp.n. is closer to the continental Pacificampa (P.birsteini, P. caesa by Chevrizov (1978) and the not-formally-described Pacificampa species by Ferguson (1997) from China, northern Korean peninsula) with some taxonomic differences in: presence of 1+1 ma macrosetae on urosternite IV, and urosternites II to VII with 4+4 macrosetae (instead of 5+5).

Discussion
Habitat and conservation issues P. daidarabotchi Sendra sp.n. was found in Mejiro-do Cave (33.763N, 130.907E) which is used for touristic purposes.This limestone cave is located in Kitakyushu City (Fukuoka Prefecture) on Kyushu Island (Fig 25A).The external environment is a typical karst landscape (Fig. 26) and is currently protected, although severe impacts occur in the surroundings.The cave entrance, as well as its first 200 meters, were adapted to the tourism, through the installation of electric lighting, pathways, stairs and banisters (Figs 27,29).This area is severely altered, and only invertebrates typically found near entrances (such as crickets and moths, for example) are observed.However, tourists do not access most parts of the cave, which presents ca.2,160 m of horizontal projection.The lower level (comprising the biggest part of the cave), is trespassed by a drainage system (Fig. 28), which brings in plant debris from the external environment, constituting the main organic resource available for the subterranean fauna.
Individuals of P. daidarabotchi Sendra sp.n were only found in deeper areas of the cave (at least 250 m far from the entrance), which are relatively well preserved (Fig. 28).Specimens were found freely walking on the muddy sediments surrounding the stream in the main conduit of the cave, over calcite deposits (speleothems -Fig.30) and sometimes under rocks (Fig. 31).Other troglobitic species found in this cave include millipedes, spiders, springtails and beetles (Stygiotrechus).However, although the cave is located within a protected area, an extensive quarry is operating in the surroundings (Fig. 32), which brings some concern regarding the protection of this species.In a straight line, the cave entrance is located less than two kms from the boundary of the quarry.Accordingly, it is advisable to start a monitoring of the cave fauna, in order to assess alterations in the communities or populations, with respect to the expansion of the quarry.Inaba-do Cave (33.439N, 133.086E) comprises a limestone cave associated to the Chichibu zone, a Jurassic accretionary prism.It is located in Tsuno Town (Kochi Prefecture), in Shikoku Island (Fig. 25B).The single entrance is located at the base of a limestone mountain and is used for touristic and religious purposes .The cave entrance region was modified to facilitate the access of humans, so that stairs and pathways were built, thus altering its pristine conditions.A stream appears (in an upwelling) in a conduit not far from the entrance, trespassing part of the cave.Although the cave is touristic, some passages are considerably narrow, thus preventing the access of visitors to the innermost portions of the cave, which are well preserved.In the deepest part of the cave there is a big chamber, formed by the collapse of rocks from the walls and ceiling, although most of the blocks are covered by a clay layer (Fig. 35).Along the conduits (especially those trespassed by the stream), there are banks of sediments, where the specimens of P. nipponica Sendra sp.n were found (Fig, 36).They were freely walking on this sediment, apparently looking for trophic resources.Although there are bats in the cave, their colonies were not big, so that guano probably does not constitute the main resource in this cave.Plant debris brought by water from external environments seems to constitute a more regular food supply for the cave fauna.Other troglobitc invertebrates observed in this cave include springtails and carabidae beetles (Ishikawatrechus cerberus).
Pacificampa nipponica Sendra sp.n. was also found in Goya Daiichi Shonyu-do cave (33.689N, 130.811E), another show cave located in the same municipality of Mejiro-do cave (in Kyushu Island -Fig.25B).It presents around 180 m of horizontal projection, but contrary to what is observed in Mejiro-do cave, practically all its extension is used for tourism.Thus, virtually the entire length of this cave is quite altered, existing pathways, stairs (Fig. 38), gates and containment structures (stone walls -Fig.37) that severely altered the pristine conditions of the cave.The trophic resources observed consisted mainly of bat guano.Specimens were observed in the final portion of the cave, under rocks present in the floor, in places further away from the areas directly trampled by tourists (Fig. 39).Considering that this cave presents uncontrolled tourism, it would be also advisable to monitor its invertebrate communities, in order to verify the effect of the tourism on the different species.

Phylogenetic affinities of the genus Pacificampa with notable biospeleological and biogeographical remarks
The characteristics of 3+3 (ma, la, lp) pronotal macrosetae and the simple claws without lateral crests put Pacificampa into Campodeinae, a subfamily widespread in all continents except in Antarctica (Conde 1956).The absence of lateral telotarsal process and the extra macrosetae on metanotum, with 2+2 lp 2,3, are not common in Campodeinae and this combination characterize Pacificampa genus, distinguishing it from other Campodeinae genera (Chevrizov 1978).These uncommon features are independently present in a few genera, such as Afrocampa Silvestri, 1933, Anisocampa Silvestri, 1932, Eumesocampa Silvestri, 1933, Mexicampa Wygodzinsky, 1944, Natalocampa Condé, 1951and Metriocampa Silvestri, 1912.These two distinctive features suggest some affinities mainly with the closely distributed genera, Eumesocampa and Metriocampa.These close affinities between Eumesocampa and Pacificampa were previously suggested by Ferguson (1997).
Eumesocampa, with three soil-dwelling species cited in several localities in the United States of North-America (Allen 1994), has a similar notal macrosetae pattern to Pacificampa, but claws show a thin spiniform lateral expansion.Furthermore, Ferguson (1982Ferguson ( , 1989) ) quoted two new undescribed species of Eumesocampa in several caves in the mid-western States.
Metriocampa, is more widespread than other genera, with 16 already described species (Allen 1994, Ferguson 1997, Chou and Chen 1980) and although in some species share the same telotarsus structure as Pacificampa, all species of Metriocampa do not have extra macrosetae in mesonotum nor metanotum.They have been found in endogean habitats from North-American localities to the east of Russia, China (Chou and Chen 1980) and in Japan (Silvestri 1931) and the most northern location of any dipluran, in Alaska (Sikes and Allen 2016).These three related genera, Eumesocampa, Metriocampa and Pacificampa, show former connection of East Asia and North-America and a result nowadays of a vicariance distribution at subfamily and genus level.
All five known Pacificampa species have been found in caves.P. birsteini, P. caesa, including one undescribed Pacificampa species from China (Ferguson 1997) are known of in three different caves in continental karstic areas; in the extreme east of Russia and in the north-east of China, all in the north of the Korean peninsula.The two new species were collected in two southern islands of Japan, Shikoku and Kyushu Islands.In the case of continental species, they have apparently no clear troglomorphic features perhaps because of the poor description of the specimens.On the contrary, P. nipponica Sendra n. sp. and P. daidarabotchi Sendra n. sp.show some troglomorphic features, more evident in P. daidarabotchi n. sp.than in P. nipponica n. sp.such as: abundant and complex sensilla of the cupuliform organ; large body with all appendages moderately elongated (Tables 1-4), elongation that is clear in antennomeres and cercal articles; and, an increase in the number of antennomeres and cercal articles compared to other soildwelling Campodeinae species and the cave-dwelling continental species of Pacificampa.
The presence of P. nipponica Sendra sp.n. in two caves on Shikoku Island and Kyushu Islands is remarkable.These two islands are now separated by a sea, but it is known that they were connected during the last glacial age (20,000-10,000 years ago).Another exemple, among the cave-dwelling fauna, that highlights the biogeographical affinity of these two islands, is that related to a couple of cryptic troglobitic beetle species of the subgenera Paratrechiama and Pilosotrechiama of the genus Rakantrechus (Trechinae) (Uéno and Naitô 2008).
P. daidarabotchi sp.n. was collected in Mejiro-do Cave, Kyushu Island, nearby the cave from which P. nipponica sp.n. was collected.A similar distribution, also dwelling in the same cave, has been reported in several subterranean campodeids; Campodea (Campodea) grassii Silvestri, 1912 and Campodea (Campodea) majorica Condé, 1955 from the extreme east of the Iberian peninsula (Sendra and Moreno 2004;D0528); Podocampa asturiana Sendra, Monedero &Salgado, 2003 andPodocampa simonini Condé, 1956 in the Cantabrian and Basque Mountains in the north of the Iberian Peninsula (Sendra et al. 2003;Sendra et al. in press) and Plusiocampa (Plusiocampa) balsani Condé, 1947 andPlusiocampa (Plusiocampa) dargilani (Moniez, 1894) in the Grands Causses south of Massif Central (Condé 1997).In such pairs of species with a sympatric distribution, the first one occupies a larger range than the second species, and the second goes much further in the troglomorphic process adaptation to the subterranean ecosystem.A remarkable fact that ought to be explored in the future.

Figure 25 .
Figure 25.Map of Japan highlighting Kyushu (A) and Shikoku (B) Islands.The red star indicates the location of Mejiro-do Cave (habitat of P. daidarabotchi), while the yellow stars indicate the location of Goya Daiichi Shonyu-do (Kyushu) and Inaba-do (Shikoku) caves, both habitats of P. nipponica.

Figures 26 -
Figures 26-31.26 Karst landscape in the area where Mejiro-do cave is located 27 Map of Mejiro-do cave, indicating the touristic area (marked in pink), and the locations where specimens of P. daidarabotchi were found (red dots) 28 conduit in the non-touristic area, trespassed by a stream 29 touristic area near the cave entrance 30 speleothems, where some specimens can be observed 31 P. daidarabotchi, living specimen.

Figure 32 .
Figure 32.Karst landscape in the area where Mejiro-do cave is located (yellow star).Below, the quarry, whose limits are less than 2km from the Mejiro-do cave entrance.

Figure 33 -
Figure 33-39.33 Karst landscape in the area where Inaba-do cave is located 34 Inaba-do cave entrance 35 chamber located at the innermost area of Inaba-do cave 36 P. nipponica, living specimen from Inaba-do cave 37 stone wall built in Goya Daiichi Shonyu-do cave 38 mettalic stairs in Goya Daiichi Shonyu-do cave 39 P. nipponica, living specimen from Goya Daiichi Shonyu-do cave.

Table 2 .
Pacificampa daidarabotchi Sendra, sp.n., cercal articles length and total length (units in mm) including number of articles of each cerci.

Table 4 .
Pacificampa nipponica Sendra, sp.n., cercal articles length and total length (units in mm) including number of articles of each cerci.