A subterranean species of Exocelina diving beetle from the Malay Peninsula filling a 4,000 km distribution gap between Melanesia and southern China

Abstract

We describe a new subterranean species of the genus Exocelina Broun, 1886 (Coleoptera: Dytiscidae) from the Malay Peninsula. Almost all of the 196 species of that genus are epigean and distributed mainly in New Guinea, Australia, Oceania and New Caledonia. One epigean species is, however, known from China. The discovery of a species on the Malay Peninsula fills that distribution gap to some degree.

Full text

New subterranean diving beetle 25
A subterranean species of Exocelina diving beetle from
the Malay Peninsula filling a 4,000 km distribution gap
between Melanesia and southern China
Michael Balke1, Ignacio Ribera2
1 SNSB-Zoologische Staatssammlung, Münchhausenstrasse 21, D-81247 München, Germany 2 Institute of
Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Barcelona, Spain
Corresponding author: Michael Balke (balke.m@snsb.de)
Academic editor: Oana Moldovan | Received 15 January 2020 | Accepted 22 February 2020 | Published 10 March2020
http://zoobank.org/E82D45EF-B6AF-4A17-831D-ED8D4DBD5A4E
Citation: Balke M, Ribera I (2020) A subterranean species of Exocelina diving beetle from the Malay Peninsula lling a
4,000 km distribution gap between Melanesia and southern China. Title. Subterranean Biology 34: 25–37. https://doi.
org/10.3897/subtbiol.34.50148
Abstract
We describe a new subterranean species of the genus Exocelina Broun, 1886 (Coleoptera: Dytiscidae)
from the Malay Peninsula. Almost all of the 196 species of that genus are epigean and distributed mainly
in New Guinea, Australia, Oceania and New Caledonia. One epigean species is, however, known from
China. e discovery of a species on the Malay Peninsula lls that distribution gap to some degree.
Keywords
Beetles, blind subterranean species, disjunct distribution, new species
Introduction
Here we report the discovery of a new subterranean diving beetle from the Malay
Peninsula. is species was placed in the Dytiscidae, subfamily Copelatinae based on
morphological characters using the key of Miller and Bergsten (2016). It was then
unambiguously assigned to the genus Exocelina Broun, 1886 in a phylogenetic analysis
using molecular systematic data of Toussaint et al. (2014, 2015, 2020 in preparation).
Subterranean Biology 34: 25–37 (2020)
doi: 10.3897/subtbiol.34.50148
http://subtbiol.pensoft.net
Copyright Michael Balke, Ignacio Ribera. 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 origina author and source are credited.
RESEARCH ARTICLE
Subterranean
Biology Published by
The International Society
for Subterranean Biology
A peer-reviewed open-access journal

Michael Balke & Ignacio Ribera / Subterranean Biology 34: 25–37 (2020)
26
e 196 described species of Exocelina are mostly from New Guinea (141 species, see
e.g. Balke 1998; Shaverdo et al. 2018, 2019; Shaverdo and Balke 2019), followed by
New Caledonia (37 species) and Australia (16 species, two of them subterranean),
with single species each in Hawaii and Vanuatu (Balke et al. 2007; Nilsson and Hájek
2019). All of these localities lie east of the Lydekkers line. A single species was discov-
ered in Shizong, Yunnan, China (Balke and Bergsten 2003), leaving a gap of around
4,000 km in the distributional range of Exocelina, essentially the entire Indonesian
Archipelago and mainland Southeast Asia. e present nding partly lls this gap and
suggests that more discoveries are to be expected, for example from the little sampled
mountain regions of Vietnam and Laos. A synopsis of the subterranean diving beetles
of the World was provided by Miller and Bergsten (2016), who provide an identica-
tion key as well as habitus photographs.
Material and methods
Specimens were studied with a Leica M205C stereo microscope at 10–160x. Images
were taken with a Canon EOS 5DS camera tted with a Mitutoyo 10x ELWD Plan
Apo objective attached to a Carl Zeiss Jena Sonnar 3.5 / 135 MC as focus lens. Illu-
mination was with two to four LED segments SN-1 from Stonemaster (https://www.
stonemaster-onlineshop.de). Image stacks were generated using the Stackmaster macro
rail (Stonemaster), and images were then assembled with the computer software Heli-
con Focus 4.77TM.
Drawings were produced with a camera lucida, rst sketched with pencil on paper,
then photographed and digitally inked using an iPad Pro and the Concepts as well as
MediBang Paint APPs.
One paratype male of the new species (voucher number IBE-AN1160) was used
for a non-destructive DNA extraction using a commercial kit (Qiagen DNeasy Tissue
Kit). We successfully amplied six mitochondrial and nuclear genes in ve sequencing
reactions, two cytochrome c oxidase subunit I fragments (COI-5’ -the "barcode"- and
COI-3’), 5’ end of rrnL RNA plus leucine tRNA transfer (tRNA-L1) plus 5’ end of
NADH dehydrogenase subunit I (NAD1), and one internal fragment of both small
ribosomal unit (18S RNA) and Histone 3 (H3) (see Villastrigo et al. 2018, for de-
tails of the primers and sequencing conditions). ese are fragments routinely used
for Dytiscidae systematics. Sequences were edited using Geneious v10.1 (Kearse et al.
2012). Here, we combined the newly obtained sequences of COI-3', 18S and H3 (ENA
database with accession numbers LR759936 H3, LR759937 18S, LR759938 3'COI,
LR760127 5'COI) with the data of Toussaint et al. (2014, 2015 as well as 2020 in
preparation). Other markers used by the latter authors (such as Carbomoylphosphate
synthase (CAD) and Alpha-Spectrin (Asp)) could not be amplied here.
e combined dataset was analysed with a fast maximum likelihood search as im-
plemented in IQ-TREE v1.6 (Nguyen et al. 2015), with a partition by gene fragment
and the best evolutionary model as selected by Modelnder (Kalyaanamoorthy et al.
2017) using the AIC (Akaike Information Criterion). We assessed topological stability

New subterranean diving beetle 27
with 1000 ultrafast bootstraps and tested tree branches by SH-like aLRT with 1000
replicates (Nguyen et al. 2015).
Repositories
IBE Institute of Evolutionary Biology, Barcelona, Spain
KSc Kazuki Sugaya collection, Zama, Japan
NMW Naturhistorisches Museum Wien, Austria
ZSM Zoologische Staatssammlung München, München, Germany
Taxonomy
Family Dytiscidae Leach, 1815
Genus Exocelina Broun, 1886
Exocelina sugayai sp. nov.
http://zoobank.org/D7A59208-6691-4E3E-8899-9942AC745D4A
Type locality. Malaysia, Pahang, Cameron Highlands, Tanah Rata, 4.474705,
101.384043.
Material examined. Holotype male (ZSM): Malaysia, Pahang, Cameron High-
lands, Tanah Rata, Mount Berembun, 4.474705, 101.384043, 1,500m, 27.–29.ii.2012,
K. Sugaya leg.
Paratypes: 4 males (1 used for DNA extraction and sequencing, voucher No. IBE-
AN1160) and 2 females, same label data as holotype (IBE, KSc, NMW, ZSM).
Description of holotype. Size and shape: Smallest Exocelina known (length of
holotype including head 2.7 mm, length without head 2.4 mm, greatest width 1.0
mm). Abdomen comparably parallel sided; pronotum also comparably parallel sided,
slightly constricted before base, hind angles produced backwards (Fig. 1A).
Coloration. Testaceous and slightly translucent (Figs 1A, B, 2A–F).
Surface sculpture. Head and pronotum with distinct microreticulation formed
by small regular cells and ne moderately dense punctation. Elytra with distinct mi-
croreticulation formed by small regular cells and dense, coarse, setiferous punctation
(Fig. 1A, D). Ventral side with distinct microreticulation formed by small regular cells,
including distinct microreticulation on metacoxal processes (Figs 3A, 4A–C).
Structures. Eyes fully reduced, with only small black scars remaining on surface
of head (Figs 1A, B, 2A, B). Male antennomeres strongly modied: 2 and 3 monili-
form, 4 slightly broadened in dorsal view, 5–11 strongly expanded, 11 at and blade
like (Fig. 1A). Fore tarsus dilated, fore angle of tarsomere 4 ventrally produced (Fig.
1C) and with two thicker setae (but no hook as in other Exocelina), on tarsomere
5 ventrally without obvious setation; pro and mesotarsomeres 1–3 with 4 rows of
stalked suction discs (2 per row). Pronotum with faint lateral bead not reaching an-

Michael Balke & Ignacio Ribera / Subterranean Biology 34: 25–37 (2020)
28
Figure 1. Exocelina sugayai sp. nov. A habitus dorsal of male B same of female C foretarsus of male, ar-
row pointing at expanded anterior ventral angle of tarsomere IV D surface sculpture on male elytral disc,
cropped from A. Length of left beetle: 2.7 mm.
terior nor posterior corners (Fig. 2B, D, F). Prosternal process short, lanceolate, de-
exed, gently rounded ventrally (Figs 3A, 4A); metaventrite broadly triangular, its
lateral “wings” very narrow (Fig. 4B, C). Membranous wings strongly reduced, with
only very short stubs visible at the wing base. Metacoxal “lines” broadly diverging,
fainting well before hind margin of metaventrite (Figs 3A, 4B). Metacoxal processes
small, more elongate oval, with wide gap in middle (to possibly enable higher mobil-
ity of hindlegs) (Figs 3A, 4B). Last ventrite apically rounded. Median lobe of aedea-
gus simply curved in lateral view, parameres of simple, Copelatinae-type triangular
shape (Fig. 5A, B).
Female. Antennomeres liform to slightly moniliform (Fig. 1B). Pro and mesotar-
someres 1–3 not bearing stalked suction discs and protarsomere 4 not modied.

New subterranean diving beetle 29
Figure 2. Exocelina sugayai sp. nov. male A eye in lateral view B detail of head and pronotum C surface
sculpture on base of head and anterior margin of pronotum D detail of posterior angle of pronotum E
detail of surface sculpture on base of elytron F detail of lateral view of elytral and pronotal base and head.

Michael Balke & Ignacio Ribera / Subterranean Biology 34: 25–37 (2020)
30
Figure 3. Ventral view of A Exocelina sugayai sp. nov. male and B Exocelina abdita.
Variation. Length of beetle including head 2.4–2.8 mm. Two paratypes are darker
orange (see Fig. 1B). According to the collector, this is due to subsequent darkening in
alcohol storage.
Etymology. Named after Kazuki Sugaya, the discoverer of this species.
Dierential diagnosis. is species diers from all other Dytiscidae by: Copelati-
nae with reduced eyes; beetle length < 3 mm; body with well visible microreticulation;
prosternal process short and deexed; metacoxal processes small, more elongate oval
(in other Copelatinae, including the groundwater species Exocelina abdita Balke et al.
2004, this structure is more rounded, and the metacoxal “lines” can be more parallel
sided, Figs 3B, 4D); male with strongly modied antennomeres.

New subterranean diving beetle 31
Figure 4. Exocelina sugayai sp. nov. male, ventral side A prosternal process and mesocoxal area B meta-
coxa and metacoxal processes C metaventrite and metaxoca D Exocelina abdita, metacoxa and metacoxal
processes. Lines in B and D inserted to highlight outline of metacoxal processes.
Habitat. Collected from two helocrenes on a slope in forested area. e beetles
were observed creeping around and were not swimming when observed (K. Sugaya
personal communication 2019) (Fig. 6A, B).
Phylogenetic anities. e best evolutionary model tting the data according to
Modelnder was a GTR+F for all partitions. Exocelina sugayai sp. nov. was recovered
deeply subordinated within Exocelina, as the sister of the Chinese E. shizong Balke &

Michael Balke & Ignacio Ribera / Subterranean Biology 34: 25–37 (2020)
32
Figure 5. Exocelina sugayai sp. nov. male genital, A median lobe of aedeagus in lateral view B paramere
lateral inner view.
Bergsten, 2003 and the New Caledonian E. nehoue Balke et al., 2014. ese three spe-
cies are part of a clade (“C4” in Toussaint et al. 2015) otherwise containing E. parvula
(Boisduval, 1835) from Hawaii as well as a clade of New Caledonian and one Vanuatu
species (Fig. 7). e other two subterranean species of Exocelina are E. abdita Balke et al.,
2004 and E. rasjadi Watts & Humphreys, 2009 from Australia. e former was included
in our phylogenetic analysis and placed in a dierent clade than Exocelina sugayai sp.
nov. (Fig. 7, included subterranean species in red). Data for E. rasjadi were not available.
Discussion
Most species of Exocelina inhabit stream associated (lotic) habitats, specically areas
of stagnant water at the edge of streams and creeks, the interstitial and tiniest of water
holes on riverbanks, as well as small puddles in intermittent creeks including the source
area that might only have occasional water ow after rainfalls (see habitat photos in
Shaverdo et al. 2012). is is the likely ancestral habitat type in Exocelina, with four
subsequent shifts to lentic habitats (and only a few species in the lentic clades) (Tous-
saint et al. 2015). Most species have limited geographic ranges; in one widespread
epigean species population genomic studies revealed strong geographic structure even
in populations as close to each other as 40 km straight line (Lam et al. 2018).

New subterranean diving beetle 33
Figure 6. Habitat of Exocelina sugayai sp. nov. A overview B detailed, with a beetle crawling about in
the center of the image.

Michael Balke & Ignacio Ribera / Subterranean Biology 34: 25–37 (2020)
34
Figure 7. Simplied phylogenetic tree obtained with IQ-TREE using the DNA sequence dataset of
Toussaint et al. (2014, 2015 as well as 2020 in preparation ) plus the newly obtained sequences of Ex-
ocelina sugayai sp. nov. Non-relevant clades are collapsed to genus or other major clades. Numbers in
nodes, ultrafast bootstrap / SH-like aLRT support.
e lotic beetles often hide in the gravel when disturbed, and observations of M.
Balke in New Guinea suggest that the interstitial of riverbanks is often utilized by these
beetles, possibly to avoid downstream drift. e beetles seem to avoid habitat with ne,
dense substrates, which we suggest make it hard to hide as such substrate clogs the
space between stones and pebbles (see also Balke 2001).

New subterranean diving beetle 35
is lifestyle could be interpreted as a preadaptation for interstitial or stygobitic
life. In fact, some Australian species seem to mainly inhabit the interstitial, and have
been suggested to provide a scenario for the transition from epigean to stygobitic life
(Watts et al. 2016). To date, two species have been described from groundwater habi-
tats in Australia. ey exhibit a strongly modied morphology typical of stygobitic
species, such as wing and eye reduction and depigmentation (Balke et al. 2004; Watts
and Humphreys 2009, see also Watts et al. 2016). e discovery of the new species
described here suggests that many more such stygobitic Exocelina could be found in
the future. Our phylogenetic analysis also suggests that the evolution of subterranean
Exocelina occurred at least two times independently (Fig. 7). In Copelatinae, one species
of the genus Copelatus Erichson, 1832 from Brazil has been described from the subter-
ranean habitat (Caetano et al. 2013).
Biogeographically, the occurrence of Southeast Asian and a Chinese species of Ex-
ocelina remains enigmatic. e origin of the clade containing these species was es-
timated as at least 10 million years ago (“C4” Toussaint et al. 2015). Based on the
information currently available, we can not state with condence whether the Asian
species are “relics” of a previously diverse and widespread Exocelina fauna, or the result
of rare dispersal events without apparent subsequent diversication.
Acknowledgements
We express our sincere thanks to Kazuki Sugaya for sending the specimens studied here
to the senior author, and Anabela Cardoso for laboratory work. Helena Shaverdo and
Günther Wewalka (Vienna) provided very valuable reviews of the submitted manu-
script. is research was supported by DFG Ba2152/4-1, 7-1, 11-1, 11-2 and 24-1.
Michael Balke acknowledges support from the EU SYNTHESYS program projects
FR-TAF-6972 and GB-TAF-6776.
References
Balke M (1998) Revision of New Guinea Copelatus Erichson, 1832 (Insecta: Coleoptera:
Dytiscidae): e running water species, Part I. Annalen des Naturhistorischen Museum
Wien 100B: 301–341.
Balke M (2001) Papuadessus pakdjoko – a new genus and species of rheobiont diving beetle
from New Guinea of potential use for environmental impact assessments (Coleoptera,
Dytiscidae). Hydrobiologia 464(1–3): 107–112.
Balke M, Bergsten J (2003) Copelatus (Papuadytes) shizong sp.n. from Yünnan (China), the rst
member of Papuadytes Balke found west of the Wallace Line. In: Jäch MA, Ji L (Eds) Water
beetles of China. Vol. 3. Zoologisch- Botanische Gesellschaft in Österreich and Wiener
Coleopterologenverein, Wien, 89–94.

Michael Balke & Ignacio Ribera / Subterranean Biology 34: 25–37 (2020)
36
Balke M, Pons J, Ribera I, Sagata K, Vogler AP (2007) Infrequent and unidirectional colonization
of hyperdiverse Papuadytes diving beetles in New Caledonia and New Guinea. Molecular
Phylogenetics and Evolution 42: 505–516. https://doi.org/10.1016/j.ympev.2006.07.019
Balke M, Watts CHS, Cooper SJB, Humphreys WF, Vogler AP (2004) A highly modied
stygobiont diving beetle of the genus Copelatus (Coleoptera, Dytiscidae): taxonomy and
cladistic analysis based on mitochondrial DNA sequences. Systematic Entomology 29:
59–67. https://doi.org/10.1111/j.1365-3113.2004.00229.x
Caetano DS, Bená DD, Vanin SA (2013) Copelatus cessaima sp. nov. (Coleoptera: Dytiscidae:
Copelatinae): rst record of a troglomorphic diving beetle from Brazil. Zootaxa 3710(3):
226 –232. https://doi.org/10.11646/zootaxa.3710.3.2
Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder:
Fast model selection for accurate phylogenetic estimates. Nature Methods 14: 587–589.
https://doi.org/10.1038/nmeth.4285
Kearse M, Moir R, Wilson A, Stones-Hava S, Cheung, Sturrock S, Buxton S, Cooper A,
Markowitz S, Duran C, ierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious
Basic: An integrated and extendable desktop software platform for the organization and
analysis of sequence data. Bioinformatics 28: 1647–1649. https://doi.org/10.1093/bioin-
formatics/bts199
Lam AW, Gueuning M, Kindler C, Van Dam M, Alvarez N, Panjaitan R, Shaverdo H, White
LT, Roderick GK, Balke M (2018) Phylogeography and population genomics of a lotic wa-
ter beetle across a complex tropical landscape. Molecular Ecology 27: 3346–3356. https://
doi.org/10.1111/mec.14796
Miller KB, Bergsten J (2016) Diving beetles of the world. Systematics and biology of the
Dytiscidae. Baltimore: Johns Hopkins University Press, 320 pp.
Nguyen L‐T, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ‐ TREE: A fast and eective
stochastic algorithm for estimating maximum likelihood phylogenies. Molecular Biology
and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
Nilsson AN, Hájek J (2019) A world catalogue of the family Dytiscidae (Coleoptera, Adepha-
ga). 307 pp. [Internet version 1.I.2019] http://www.waterbeetles.eu
Shaverdo H, Balke M (2019) A new species of the Exocelina ekari group and new faunistic
data on 12 species of Exocelina Broun, 1886 from New Guinea (Coleoptera: Dytiscidae).
Koleopterologische Rundschau 89: 1–10.
Shaverdo H, Sagata K, Balke M (2018) Introduction of the Exocelina casuarina-group, with a
key to its representatives and descriptions of 19 new species from New Guinea (Coleoptera,
Dytiscidae, Copelatinae). ZooKeys 803: 7 –70. https://doi.org/10.3897/zookeys.803.28903
Shaverdo HV, Surbakti S, Hendrich L, Balke M (2012) Introduction of the Exocelina ekari-
group with descriptions of 22 new species from New Guinea (Coleoptera, Dytiscidae,
Copelatinae). ZooKeys 250: 1–76. https://doi.org/10.3897/zookeys.250.3715
Shaverdo HV, Surbakti, Warikar EL, Sagata K, Balke M (2019) Nine new species groups, 15
new species, and one new subspecies of New Guinea diving beetles of the genus Exocelina
Broun, 1886 (Coleoptera, Dytiscidae, Copelatinae). ZooKeys 878: 73–143. https://doi.
org/10.3897/zookeys.878.37403

New subterranean diving beetle 37
Toussaint EFA, Hall R, Monaghan MT, Sagata K, Ibalim S, Shaverdo HV, Vogler AP, Pons J,
Balke M (2014) e towering orogeny of New Guinea as a trigger for arthropod megad-
iversity. Nature Communications 5: 4001. https://doi.org/10.1038/ncomms5001
Toussaint EFA, Henrich L, Shaverdo H, Balke M (2015) Mosaic patterns of diversication
dynamics following the colonization of Melanesian islands. Scientic Reports 5: 16016.
https://doi.org/10.1038/srep16016
Villastrigo A, Fery H, Manuel M, Millán A, Ribera I (2018) Evolution of salinity tolerance in
the diving beetle tribe Hygrotini (Coleoptera, Dytiscidae). Zoologica Scripta 47: 63–71.
https://doi.org/10.1111/zsc.12255
Watts CHS, Humphreys WF (2009) Fourteen new Dytiscidae (Coleoptera) of the genera Lim-
bodessus Guignot, Paroster Sharp, and Exocelina Broun from underground waters in Aus-
tralia. Transactions of the Royal Society of South Australia 133(1): 62–107. https://doi.or
g/10.1080/03721426.2009.10887112
Watts CHS, Hendrich L, Balke M (2016) 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). Subterranean Biology 19: 23–29. https://doi.
org/10.3897/subtbiol.19.9513