A new interstitial species of diving beetle from tropical northern Australia provides a scenario for the transition of epigean to stygobitic life (Coleoptera, Dytiscidae, Copelatinae)

Exocelina saltusholmesensis sp. n. is described from a single female collected in Holmes Jungle Reserve near Darwin in the Northern Territory, Australia. Phylogenetically, the new species is sister to a clade containing the epigean E. ferruginea (Sharp, 1882) and E. punctipennis (Lea, 1899) but well characterized by its smaller size, the much smaller eyes, vestigial wings and paler surface. Exocelina saltusholmesensis sp. n. was collected from a small pool in an intermittent and temporary small creek. The collecting circumstances suggest that this is an interstitial species, with morphological characters interpreted as adaptations to a strongly hidden if not mostly subterranean lifestyle.

A new interstitial species of diving beetle from tropical northern Australia provides a scenario for the transition of epigean to stygobitic life (Coleoptera, Dytiscidae, Copelatinae)

Introduction
The diving beetle genus Exocelina contains more than 140 described species, most of them in New Guinea and New Caledonia (Toussaint et al. 2014(Toussaint et al. , 2015;;Nilsson 2015).The majority of species occur in habitats associated with streams, but there were also four transitions to stagnant water habitats (Toussaint et al. 2015).Two highly derived stygobitic Australian species have been described to date, Exocelina abdita Balke et al. 2004 from Newhaven Station, Camel Bore, Northern Territory and E. rasjadi Watts & Humphreys, 2009 from Tjukurla calcrete, Lake Hopkins palaeovalley, Western Australia.Both species are blind (Fig. 2B, 3C), have vestigial wings, are depigmented and show a more or less pronounced constriction between base of pronotum and elytra (Fig. 2B) which is linked to reduction of flight muscles (Balke et al. 2004, Watts andHumphreys 2009).Another rarely collected Australian species, Exocelina australis (Clark, 1863), has smaller eyes than typical epigean species and is only lightly pigmented (Fig 2A).This might indicate a hidden, interstitial lifestyle.The collection of the species from hyporheic environments of a number of localities in the Flinders Range also hints in this direction (Leys et al. 2010).
Here, we describe a new species collected from a small pool in the bed of a slowly flowing wet-season creek near Darwin.Morphological features such as reduced eyes and vestigal wings, light pigmentation and absence of the species from other nearby water bodies suggest this is an interstitial species providing a scenario for the transition from epigean to stygobitic life.This discovery highlights the hidden diversity of an underexplored habitat.

Methods
Morphological observations were made and measurements were taken with a Leica M205 C stereomicroscope.Specimens were illuminated by a diffuse 13W fluorescent double light tube.
A DNA sequence of the 3' fragment of the mitochondrial cytochrome oxidase 1 gene was generated by Remko Leys at the South Australian Museum in Adelaide, using the oligonucleotide primers Jerry and Pat (TCC AAT GCA CTA ATC TGC CAT ATT A / CAA CAT TTA TTT TGA TTT TTT GG).
Digital images were taken with a Nikon D3X equipped with a bellow or expansion rings and lenses: Mitutoyo 10x ELWD Planapo or Leitz Photar 25/2.8.Illumination came from three compact Nikon flashes, and the instrument was moved on an Isel linear drive (https://www.isel.com/at/lineareinheiten-les6.html) or Cognisys Stackshot (for very small steps 1-7 µm) (www.cognisys-inc.com).Image stacks were combined using the method A in Helicon focus software (www.heliconsoft.com).European nucleotide archive (genbank) accession number for the 3' end of the mitochondrial cytochrome c oxidase 1 gene: www.ebi.ac.uk/ena/data/view/LN994839

Family
Description.Size and shape: Beetle small (length of beetle 3.7 mm, greatest width 1.8 mm, length of pronotum 0.6 mm, width of pronotum at base 1.7 mm), comparably parallel-sided (Fig. 1C).
Structures.Eyes small in comparison to epigean Exocelina, length of eye c. 0.16 mm, distance between eyes c. 0.86 mm (Figs 1C, 3B).Pronotum with faint lateral bead that does not reach anterior corner.Prosternal process lanceolate, gently rounded ventrally; lateral wings of metaventrite narrow.Flight wings vestigial. Male.Unknown.
Etymology.The name is an adjective is the nominative singular derived from the locality name -Holmes Jungle.Latin: saltus = woodland.
Differential diagnosis.The species is sister to a clade containing Exocelina punctipennis (Lea, 1899) and E. ferruginea (Sharp, 1882) established based on DNA sequence data (Toussaint et al. 2015), but both these species are larger (> 5mm), and have larger eyes (Figs 1A-C, 3A, B).Both are epigean species, distributed along the eastcoast of Queensland (E.punctipennis) and the Adelaide Hills in South Australia (E. ferruginea) (Hendrich et al. in prep.).They are also fully winged.
Habitat.A small pool in the bed of a small ephemeral creek through eucalypt woodland.A small eyed, wingless Hydraenidae (Hydraena wattsi Perkins, 2007) was collected at the same time (Perkins 2007).During the dry season (June-September) this creek dries out.However, we assume that subsurface water remains in the watercourse during this season providing an interstitial habitat for these flightless, low vagility species.

Discussion
A number of West Palearctic diving beetle species are known from wells or from the interstitial of gravel banks along rivers.These were classified as semi-subterranean or interstitial species.Morphological characters hinting at this include the reduction of eyes, depigmentation, presence of long sensory setae and the reduction of wings (e.g.Fery et al. 2012;Hernando et al. 2012;Manuel 2013).
Here we add another candidate from interstitial fauna that might document the first steps into fully underground habitats and highlights the need for focused investigations of such habitats in Australia and beyond.