Biodiversity Data Journal 9: e59892 OO)
doi: 10.3897/BDJ.9.e59892 open access
Taxonomic Paper

Hydraena (s.str.) dinarica, new species
(Coleoptera: Hydraenidae) along with further
records of Hydraena spp. from Durmitor National
Park, Montenegro and comments on the DNA
barcoding problem with the genus

Hendrik Freitag*, Rick de Vries§, Marta Paterno!, Simone Maestri!, Massimo Delledonnel,
Cameron G Thompson", Helena Lamed*, Rebekah Lambert”, Michael F Fox", Mariela C Gonzalez‘,
Emmanuel D Delocado*, Marc R Sabordo?, Clister V Pangantihon#, Ilva Njunjic“”

+ Ateneo de Manila University, Quezon City, Philippines

§ Amstelveenseweg 980B, Amsterdam, Netherlands

| University of Verona, Department of Biotechnology, Strada Le Grazie 15, Verona, Italy

| Oxford Brookes University, Department of Health and Life Sciences, Oxford, United Kingdom

# 39 av Melbourne, Mont-Royal, Quebec, Canada

= 340 N. Adams Street, Eugene, Oregon, United States of America

« Taxon Expeditions, Rembrandtstraat 20, Leiden, Netherlands
» Naturalis Biodiversity Center, Darwinweg 2, Leiden, Netherlands

Corresponding author: Hendrik Freitag (hfreitag@ateneo.edu)

Academic editor: David Bilton

Received: 21 Oct 2020 | Accepted: 28 Dec 2020 | Published: 12 Jan 2021

Citation: Freitag H, de Vries R, Paterno M, Maestri S, Delledonne M, Thompson CG, Lamed H, Lambert R, Fox
MF, Gonzalez MC, Delocado ED, Sabordo MR, Pangantihon CV, Njunjié | (2021) Hydraena (s.str.) dinarica, new

species (Coleoptera: Hydraenidae) along with further records of Hydraena spp. from Durmitor National Park,
Montenegro and comments on the DNA barcoding problem with the genus. Biodiversity Data Journal 9: e59892.

https://doi.org/10.3897/BDJ.9.e59892
ZooBank: urn:lsid:zoobank.org:pub:5c993a9a-b6ae-42ad-b349-4f995b757bde

© Freitag H 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 Freitag H et al

Abstract
Background

Long-palped Water Beetles were collected during a taxon expedition in Montenegro which
involved citizen scientists, students and taxonomists. The material was collected from
springs, brooks, fens and the Tara River, at altitudes between 600 m and 1450 m above
sea level, using fine-meshed hand-nets and by manual checking of submerged substrates.
The morphological species delimitation was supplemented and congruent with mtDNA
sequences mainly obtained in the field using the newly-developed MinlION-based ON Track
pipeline.

New information

The new species Hydraena dinarica Freitag & de Vries, sp. n. from Durmitor Mt. is
described, illustrated and compared in detail to closely-related congeners of the H. saga
d'Orchymont, 1930/H. emarginata Rey, 1885 species complex. Five additional species and
female specimens of two unidentified morphospecies of the genus were also recorded in
the vicinity of Durmitor National Park. New records and the first DNA barcodes for
Hydraena biltoni Jach & Diaz, 2012 (endemic to Montenegro) and H. morio Kiesenwetter,
1849 are provided. Further records of H. nigrita Germar, 1824, H. minutissima Stephens,
1829, H. subintegra Ganglbauer, 1901 and females of two unidentified morphospecies are
commented upon. The resulting inter- and intraspecific genetic distances and some
observations of low or zero sequence divergence between recently-diverged species of
Hydraena Kugelann, 1794 are briefly discussed.

Keywords

citizen science, MinlION sequencing, minute moss beetle, Palaearctic region, Taxon
Expeditions, Durmitor Mt.

Introduction

The Long-palped Water Beetles of the genus Hydraena, originally described by Kugelann
(1794), represent the most speciose aquatic coleopteran genus. In tropical Asia, where the
genus is still under-explored, a new species has even been discovered in the middle of a
megacity (Freitag 2013). Nineteen Hydraena species are currently recorded from
Montenegro. Many species of the genus are endemic to comparably small distribution
ranges (Jach et al. 2005), such as H. biltoni Jach & Diaz, 2012 and H. latebricola Jach,
1986 in Montenegro (see Jach 1986, Jach and Diaz 2012). The Mediterranean area is
particularly diverse and new species are still being discovered (e.g. Bilton 2013, Micetic
Stankovic and Jach 2012, Jach and Diaz 2012). Therefore, the first author targeted
Hydraena when instructing a citizen scientist project during a “taxon expedition” to

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 3

Montenegro in 2019. The principles and great benefit of such initiatives are discussed by
Schilthuizen et al. (2017) and Freitag et al. (2018). Through the enthusiastic support of
citizen scientists, several aquatic habitats in and around Durmitor National Park, a
UNESCO world heritage site, were sampled for aquatic beetles. The variety of collection
sites included springs, creeks, lakes and fens in forests and alpine meadows, up to the
stunning torrent of the Tara River in Europe's deepest gorge.

The subsequent identification of the collected material at the improvised field laboratory
(Fig. 1, Suppl. material 2), set up in a holiday resort “Etno selo Slieme” near the town of
Zabljak, revealed eight Hydraena (s.str.) species, amongst which one new species was
actually discovered. The new species was found at Skakala stream, a mountain creek
flowing from Skakala waterfall into the periodically-inundated Susica Lake on the northern
slopes of Durmitor massif (Fig. 2).

Figure 1. EES]

Citizen scientists working in an improvised lab during the taxon expedition to Durmitor.

Figure 2. EES
Skakala stream at the northern slopes of Durmitor massif, type locality of Hydraena dinarica,
sp. n.

4 Freitag H et al

Materials and methods

Specimens were collected in a microhabitat-specific approach (Freitag 2015) by disturbing
the submerged substrates of the water body and collecting any floating specimens using
fine-meshed hand-nets. Larger solid substrates (e.g. submerged wood) were taken off from
the water and checked for specimens. The material was immediately preserved in vials
with 96% ethanol, separately for each microhabitat assessed.

Pre-sorting and genus-level identification were performed by taxon expedition participants
mentored by the first author (Fig. 1), using taxonomic literature and dissection
microscopes. Specimens of Hydraena were then dissected by the help of fine pincers and
entomological pins to reveal their most diagnostic sexual characters (aedeagus,
gonocoxite and tergite X) and compared with descriptions of Hydraena species of the
Balkan Region. Their genitalia, temporarily mounted in lactic acid on microscopic slides,
were examined under a Leica ICC50 HD compound microscope.

Detailed examination and digital imaging of dissected parts was done using an Olympus
CX21 microscope equipped with a DinoEye Eyepiece camera. Habitus photographs were
taken under a Zeiss Axio Zoom V 16 microscope with a Canon 5D Mark II SLR attached to
the microscope. Images were captured at various focus planes and subsequently stacked
using the Helicon Focus software. Genital drawings were compiled after their photographs
by vector graphic tools in CorelDRAW v.10.0 software, but in direct comparison with the
actual genitals mounted on slides.

After removal of diagnostic parts (glued on entomological cards), the entire remaining
specimen of each initially-recognised morphospecies and some unidentifiable female
specimens underwent DNA isolation, amplification, sequencing and processing of the 5’-
end of the mitochondrial cytochrome oxidase | (CO/) gene as described in Maestri et al.
(2019) (see hitps://github.com/MaestSi/ONTrack). The primer pair LCO1490 and HC02198
was used for PCR amplification. Library preparation for the MinION Oxford Nanopore NGS
device was performed using SQK-LSK109 (Run1) and SQK-LSK108 (Run2) kits and, for
each library, samples were pooled together after adding index sequences. The final
libraries were loaded on a R9.4.1 Flongle flow-cell (Run1) and on a R9.4.1 MinlON flow-
cell (Run2). Sequencing was carried out in the field using an off-line version of MinKNOW
v1.6.11. The two sequencing runs were stopped after 5 and 17 hours and produced a total
of 182,504 and 533,919 sequence reads, respectively. Sequence reads were base-called
and demultiplexed using Guppy v.3.1.5 and accurate consensus sequences were
generated using the ONTrack pipeline v.1.2.2 (Maestri et al. 2019). After our return from
the field expedition, some additional sequences were generated by conventional Sanger
sequencing by a commercial service using PCR products amplified as described above
and following standard protocols. Forward and reverse Sanger reads were manually
assembled into a consensus sequence using BIOEDIT version 7.2.5 (Hall 1999).

The DNA sequence alignment, which also included available standard barcodes of related
Hydraena species of the region of Hendrich et al. (2015), Pentinsaari et al. (2014), Ribera

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 5

et al. (2011), Rulik et al. (2017), Trizzino et al. (2011) and Trizzino et al. (2013b), was
manually generated with the software MEGA X (Kumar et al. 2018).

A statistical parsimony haplotype network was constructed by TCS1.21 (Clement et al.
2002), visualised using POPART (Leigh and Bryant 2015) and further edited in Adobe
Illustrator 2020. The genetic sequence divergence analysis was performed in MEGA X
using Kimura-2-parameter (K2P) model with the bootstrap method in 1000 replicates.

DNA sequences were submitted to International Nucleotide Sequence Database
Collaboration (INSDC) through GenBank, as well as to the Barcode of Life Data System
(BOLD) under project [XEX.

The type labels of the new species are literally quoted from the specimen’s label under
‘pibliographicCitation’. Back slashes indicate the next line in the label.

Taxon treatments

Hydraena (Hydraena) biltoni Jach & Diaz, 2012

Material

a. location|ID: MNE21c; continent: Europe; waterBody: small Black Lake tributary; country:
MONTENEGRO; municipality: Zabljak; locality: Durmitor National Park; verbatimLocality:
small Black Lake tributary creek near war monument, pine forest, pebble in shallow run;
verbatimElevation: 1435 m; verbatimCoordinates: 43 08 57N; 19 05 42E; eventDate:
2019-07-16; individuallD: H69; sex: 3 males, 2 females; lifeStage: adults; recordedBy:
Hendrik Freitag, Michael F. Fox, Rebekah Lambert; associatedSequences: GenBank:
MT784158.1; identifiedBy: Hendrik Freitag, Rick De Vries, Cameron G. Thompson,
Helena Lamed, Rebekah Lambert, Michael F. Fox, Mariela Gonzalez, Clister V.
Pangantihon; institutionCode: CFM, ZMB; collectionCode: Coleoptera; basisOfRecord:
Dried specimens; informationWithheld: MONTENEGRO: Zabljak, Durmitor N.P., small
Black Lake tributary creek near war monument, pine forest, pebble in shallow run, 1435
ma.s.l., 43 08 57N 19 05 42E, 16 July 2019, leg M.F. Fox, R. Lambert, H. Freitag
(MNE21c)

Distribution

Hydraena biltoni (Fig. 3A) is endemic to Montenegro. Previously, it was collected from
the vicinity of Savnik, about 20 km south of Zabljak and Biogradska Gora (Jach and
Diaz 2012).

Remarks

We provide the first standard barcode for the species. It varies by only 0.3% from that
of the closest congener H. morio Kiesenwetter, 1849 (Suppl. material 1). As H. biltoni is
extremely similar to the latter, which also occurs in the region, thorough examination of
the aedeagus is required for proper identification.

6 Freitag H etal
D

:
C
E F
Figure 3. EESl

Digital habitus photograph of A) Hydraena biltoni 3; B) H. minutissima 3; C) H. morio 9; D) H.
nigrita 4; E) H. subintegra, pale colouration of a teneral ~ specimen; F) H. subintegra 2,
regular colouration; scale bar = 1.0 mm.

Habitat

The specimens were collected in a very small creek, flowing through pine forest and a
wet meadow. Bottom pebbles, mixed with CPOM, in moderately fast flowing, shallow
portions of the creek, were their microhabitat.

Hydraena (Hydraena) minutissima Stephens, 1829

Material

a. location|ID: MNE17b; continent: Europe; waterBody: Shallow littoral, pool with pebbles;
country: Montenegro; municipality: Zabljak; locality: Tara River; verbatimLocality: Tara
River, near Bijela Stijena, shallow littoral pool with pebbles; verbatimElevation: 600 m;
verbatimCoordinates: 43 13 23N; 19 09 57E; eventDate: 2019-07-12; individuallD: H60;
sex: 2 males, 5 females; lifeStage: adults; recordedBy: Hendrik Freitag, Clister V.
Pangantihon; associatedSequences: GenBank: MT784159.; identifiedBy: Hendrik Freitag,
Rick De Vries, Cameron G. Thompson, Helena Lamed, Rebekah Lambert, Michael F.
Fox, Mariela Gonzalez, Clister V. Pangantihon; institutionCode: CFM, ZMB;
collectionCode: Coleoptera; basisOfRecord: Dried specimens

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 7

Distribution

Hydraena minutissima (Fig. 3B), originally described from Great Britain (Stephens
1829), is widely distributed in southern, western and central Europe from Spain and
Turkey in the south up to the British Isles (Jach and Skale 2015).

Habitat

The species was collected in the shallow littoral of the Tara River with pebble deposits
on bedrock. The microhabitat was not exposed to strong currents during the time of
collection and filamentous algae were partly growing on the surrounding exposed
rocks.

Hydraena morio Kiesenwetter, 1849

Material

a. location|ID: MNE17b; continent: Europe; waterBody: Small littoral pool with pebbles;

country: Montenegro; municipality: Zabljak; locality: Tara River; verbatimLocality: Tara
River, near Bijela Stijena, shallow littoral pool with pebbles; verbatimElevation: 600 m;
verbatimCoordinates: 43 13 23N; 19 09 57E; eventDate: 2019-07-12; individuallD: H62,
H64, H70, H71; sex: 7 males, 2 females; lifeStage: adults; recordedBy: Hendrik Freitag,
Clister V. Pangantihon; associatedSequences: GenBank: MT784154.1, MT784155.1,
MT784156.1, MT784157.1; identifiedBy: Hendrik Freitag, Rick De Vries, Cameron G.
Thompson, Helena Lamed, Rebekah Lambert, Michael F. Fox, Mariela Gonzalez, Clister
V. Pangantihon; collectionCode: Coleoptera; basisOfRecord: Dried specimen

Distribution

Hydraena morio (Fig. 3C) is mainly distributed in eastern and central Europe in an area
from Turkey to Germany, including the Balkan Region (Jach and Skale 2015).

Remarks

We provide here the first CO/ 5'-end sequences (Folmer Region) of the species. See
also remarks on H. biltoni.

Habitat

For notes on the habitat, see Hydraena minutissima.

Hydraena (Hydraena) nigrita Germar, 1824

Material

a. locationID: MNE17b; continent: Europe; waterBody: Shallow littoral pool with pebbles;
country: Montenegro; municipality: Zabljak; locality: Tara River; verbatimLocality: Tara
River, near Bijela Stijena, shallow littoral pool with pebbles; verbatimElevation: 600 m;
verbatimCoordinates: 43 13 23N; 19 09 57E; eventDate: 2019-07-12; individuallD: H61;

8 Freitag H etal

sex: 2 males; lifeStage: adults; recordedBy: Hendrik Freitag, Clister V. Pangantihon;
associatedSequences: GenBank: MT784150.1; identifiedBy: Hendrik Freitag, Rick De
Vries, Cameron G. Thompson, Helena Lamed, Rebekah Lambert, Michael F. Fox, Mariela
Gonzalez, Clister V. Pangantiho; institutionCode: CFM, ZMB; collectionCode: Coleoptera;
basisOfRecord: Dried specimen

Distribution

Hydraena _ nigrita (Fig. 3D), originally described from Germany (Germar 1824), is
distributed from Greece and Eastern Europe up to the British Isles (Jach and Skale
2015).

Habitat

For notes on the habitat, see Hydraena minutissima.

Hydraena (Hydraena) spp.

Materials

a. location|ID: MNE10I; continent: Europe; waterBody: Fen meadow near Durmitor N.P
entrance; country: Montenegro; municipality: Zabljak; locality: Durmitor N.P.;
verbatimLocality: Fen meadow near Durmitor N.P entrance, littoral pool:
verbatimElevation: 1425 m; verbatimCoordinates: 43 06 08N; 19 10 56E; eventDate:
2019-07-10; individuallD: H65; sex: 3 females; lifeStage: adults; recordedBy: Hendrik
Freitag; associatedSequences: GenBank: MT784151.1; identifiedBy: Hendrik Freitag,
Rick De Vries, Cameron G. Thompson, Helena Lamed, Rebekah Lambert, Michael F.
Fox, Mariela Gonzalez, Clister V. Pangantihon; identificationRemarks: “cf. britten?’;
institutionCode: CFM; collectionCode: Coleoptera; basisOfRecord: Dried specimen

b. locationID: MNE13k; continent: Europe; waterBody: fen meadow near Dobri Nugo;
country: Montenegro; municipality: Zabljak; locality: Dobri Nugo; verbatimLocality: Fen
meadow near Dobri Nugo, partly subterranean water flow, slow run; verbatimElevation:
1392 m; verbatimCoordinates: 43 09 15N; 19 06 06E; eventDate: 2019-07-11;
individuallD: H66; sex: 2 females; lifeStage: adults; recordedBy: Hendrik Freitag;
associatedSequences: GenBank: MT784152.1; identifiedBy: Hendrik Freitag, Rick De
Vries, Cameron G. Thompson, Helena Lamed, Rebekah Lambert, Michael F. Fox, Mariela
Gonzalez, Clister V. Pangantihon; identificationRemarks: "sp. female"; institutionCode:
CFM; collectionCode: Coleoptera; basisOfRecord: Dried specimen

Remarks

The samples cluster with Hydraena britteni Joy, 1907, originally described from
England and Ireland (Joy 1907), vary only by ca. 0.3% and 2.5%, respectively, in their
genetic distance from the latter. Based on the known distribution range of H. britteni,
which does not include Montenegro and the genetic distance, it remains uncertain if
either specimens are conspecific with the latter. Due to the lack of male specimens, we
currently cannot identify these specimens with certainty.

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 9

Habitat

The specimens were collected from fen-like meadows, one (MNE10) densely
vegetated with sedge and horsetail, the other (MNE13) additionally with limestone
boulders and gravel densely covered with mosses. In both sites, a creek with clear
brownish water, rich in humins, was passing the fens and provides continuous water
inputs.

Hydraena subintegra Ganglbauer, 1901

Materials

a. location|ID: MNE17c; continent: Europe; waterBody: Black Lake tributary creek; country:
Montenegro; municipality: Zabljak; locality: Durmitor N.P.; verbatimLocality: Tara River
near Bijela Stijena, littoral, run with pebble; verbatimElevation: 600 m;
verbatimCoordinates: 43 13 23N; 19 09 57E; eventDate: 2019-07-12; individuallD: H63;
sex: 1 male, 1 female; lifeStage: adults; recordedBy: Hendrik Freitag & Clister
Pangantihon; associatedSequences: GenBank: MT784149.; identifiedBy: Hendrik Freitag,
Rick De Vries, Cameron G. Thompson, Helena Lamed, Rebekah Lambert, Michael F.
Fox, Mariela Gonzalez, Clister V. Pangantihon; institutionCode: CFM; collectionCode:
Coleoptera; basisOfRecord: Dried specimens

b. location!ID: MNE20c/f/h; continent: Europe; waterBody: Black Lake tributary creek;
country: Montenegro; municipality: Zabljak; locality: Durmitor N.P.; verbatimLocality: Black
Lake tributary creek near old watermill, pine forest; verbatimElevation: 1450 m;
verbatimCoordinates: 43 09 O9N; 19 05 22E; eventDate: 2019-07-16; individuallD: H63;
sex: 1 male, 1 female; lifeStage: adults; recordedBy: Helena Lamed, Mariela Gonzales,
Rebekah Lambert, Michael F. Fox, Clister V. Pangantihon; identifiedBy: Hendrik Freitag,
Rick De Vries, Cameron G. Thompson, Helena Lamed, Rebekah Lambert, Michael F.
Fox, Mariela Gonzalez, Clister V. Pangantihon; institutionCode: NMW, ZMB;
collectionCode: Coleoptera; basisOfRecord: Dried specimens

Distribution

The species is distributed in an area between the Adriatic and Black Seas, including
the Dinaric Alps (Jach and Skale 2015).

Remarks

The taxonomy of this species of the "Haenydra" lineage is not yet finally resolved.
Three slightly varying morphs are recognised. Our specimens (Fig. 3E, F) belong
morphologically and geographically to “Morph A” sensu Jach and Diaz (2012). The
standard DNA barcode of this morph which we are providing herein varies, in fact, by
0.5% from “Morph B” (Bulgaria).

Habitat

All specimens were collected from moderately fast flowing, shallow water, but on
varying substrates, including submerged wood, grass bunches and pebble.

10

Freitag H et al

Hydraena (Hydraena) dinarica Freitag & de Vries, sp. n.

ZooBank urn:lsid:zoobank.org:act:E5A0DED7-16AC-4B94-B9B1-72000874197D
Barcode of Life TXEX049-20

Materials

Holotype:

a.

locationID: MNE18; continent: Europe; waterBody: Skakala stream; country: Montenegro;
municipality: Zabljak; locality: Durmitor, Peradova gora; verbatimElevation: 1220 m;
locationRemarks: cold water karst creek with predominant flow subsurface;
verbatimCoordinates: 43 09 54N; 18 59 59E; samplingProtoco!: Manual collection by
hand-net from bottom substrates; eventDate: 2019-07-13; sex: male; lifeStage: adult;
recordedBy: Hendrik Freitag, Clister V. Pangantihon; identifiedBy: Hendrik Freitag; type:
Dried specimen; bibliographicCitation: MONTENEGRO: Durmitor, Peradova gora, \
Skakala stream, ca. 1220 m asl., \43°09'54"N 18°59'59"E, 13 July 2019 \ leg. H. Freitag
& C.V. Pangantihon (MNE18); institutionCode: NMW; basisOfRecord: Dried specimen;
informationVWithheld: Terminal parts of abdomen, aedeagus and right foretasus (broken
off) glued separately on to same entomological card along with holotype specimen.

Paratypes:

a.

locationID: MNE18; continent: Europe; waterBody: Skakala stream; country: Montenegro;
municipality: Zabljak; locality: Durmitor, Peradova gora; verbatimElevation: 1220 m;
locationRemarks: cold water karst creek with predominant flow subsurface;
verbatimCoordinates: 43 09 54N; 18 59 59E; samplingProtocol!: Manual collection by
hand-net from bottom substrates; eventDate: 2019-07-13; individual!D: H67, H68; sex: 14
males, 11 females; lifeStage: adults; recordedBy: Hendrik Freitag, Clister V. Pangantihon;
associatedSequences: GenBank: MT784148.1; identifiedBy: Hendrik Freitag, Rick De
Vries, Cameron G. Thompson, Helena Lamed, Rebekah Lambert, Michael F. Fox, Mariela
Gonzalez, Clister V. Pangantihon; type: Dried specimens; bibliographicCitation:
MONTENEGRO: Durmitor, Peradova gora, \ Skakala stream, ca. 1220 m asl., \
43°09'54"N 18°59'59"E, 13 July 2019 \ leg. H. Freitag & C.V. Pangantihon (MNE18);
institutionCode: CFM, NMW, SMTD, ZMB; collectionCode: Coleoptera

continent: Europe; country: Montenegro; municipality: Zabljak; locality: Durmitor, Zeleni
Vir; samplingProtocol: Manual collection; eventDate: 2002-08-02; sex: 1 female; lifeStage:
adult; recordedBy: Vladimir PeSic¢; identifiedBy: Manfred A. Jach; type: Dried specimen;
institutionCode: NMW; collectionCode: Coleoptera

Description

Habitus as in Fig. 4. Body (labrum to elytral apex) 2.25-2.45 mm long, 0.81—0.86 mm
wide. Head, pronotum and elytra dark brown to black, femora and tibiae slightly paler
dark brown, palpi and tarsi yellowish-brown. Labrum densely micropunctate, with deep
anterior notch; margins slightly upturned. Clypeus medially densely micropunctate,
gradually more microstriate laterad. Fronto-clypeal suture bisinuately arched, slightly
impressed. Frons medially moderately densely punctate; interstices glabrous; lateral
portions densely (sometimes rugosely) bipunctate; micropunctures very dense;
interocular grooves indiscernible. Eyes moderately large, distinctly protruding, about 30
facets visible in dorsal view. Maxillary palpi about as long as body width.

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 11

Figure 4. EES]

Digital habitus photographs of Hydraena dinarica, sp. n., paratype 3; scale bar = 1.0 mm.

Pronotum broadly subhexagonal, moderately wider than long; anterior and posterior
margins slightly concave; anterior and posterior angles bluntly rounded, lateral rim
denticulate, most conspicuous anteriorly; disc slightly convex; sagittal, anterior and
posterior portions densely punctate; remaining disc portions moderately densely
punctate; interstices glabrous; anterior and posterior sublateral foveae_ slightly
impressed, rather inconspicuous; entire lateral portions slightly deflexed, rugulously
bipunctate, partly microstriate.

Elytra elongate, almost parallel-sided apical 0.15—0.70; disc slightly vaulted,
sublaterally more abruptly declivitous; elytral margin moderately explanate up to ca.
apical 0.15. Elytra with six regularly arranged, not or slightly impressed rows of
puncture striae between suture and disc declivity (approx. at the middle of shoulder)
and ca. six additional, less regular puncture striae between disc declivity and elytral
margin; punctures moderately large and moderately deeply impressed on anterior disc,
gradually slightly decreasing in size and degree of impression towards apex and
margin; intervals and interstices flat and glabrous; intervals smaller than puncture
diameter anteriorly, larger in posterior and lateral portions; apical sutural teeth present
or absent, apices separately rounded, sexually dimorphic (Fig. 5B, C).

Ventral side as in Fig. 5A. Mentum and submentum densely micropunctate. Genae and
gula dominantly micropunctate, partly striate; posterior genal ridge distinct, glabrous.
Hypomeron micropunctate to microreticulate. Prosternum densely micropunctate with
hydrofuge micropubescence, with conspicuous median keel. Mesoventrite densely
micropunctate with hydrofuge micropubescence, deeply impressed anterior to
mesocoxae; impression transverse-arcuate; with pair of posteriad divergent glabrous
streaks lateral to mesocoxae, across mesoventral impression (rather inconspicuous in
some specimens); mesoventral disc and process convex. Metaventrite densely
micropunctate with hydrofuge micropubescence, central disc shallowly impressed;

12

Freitag H et al

metaventral plaques distinct, divergent posteriad; intermetacoxal process declined
posteriad, predominantly glabrous. Pseudepipleuron longitudinally impressed, with one
indistinct puncture stria, rugulose and most anteriorly with hydrofuge pubescence,
increasingly glabrous posteriad. Ventrites I-IV (externally visible sternites III—VI)
medially flat and with moderately long pubescence (most conspicuous in young
specimens), remaining portions with dense, short pubescence; ventrites V and VI
largely non-pubescent, glabrous to reticulate.

Figure 5. EES]

Hydraena dinarica, sp. n., paratypes: A) @ ventral view; B) ¢ elytral apices; C) @ elytral
apices; D) 2 ventral abdomen; E) ° tergite X; F) gonocoxite; G) 4 mesotibia; H) 2 mesotibia;
|) 4 metatibia; J) 9 metatibia.

Male terminal sternite subsemicircular, 0.18 mm wide, not distinguishable from H. saga
and similar species (comp. Jach and Diaz (2017): Figs. 5, 10 and 15); spiculum slightly
curved in apical half, 0.70-0.73 mm long (vs. ca. 0.75 mm in H. saga; comp. Jach and
Diaz (2017): Fig. 10).

Aedeagus (Fig. 6): Total length ca. 780 um; main piece (630 um long) with three long
setae on inner (left) side and a very short one on outer (right) side; apex somewhat
variable from convex to obliquely truncate, narrowest subapically; dorsal corner very
slightly produced; main piece moderately slender, basal portion rectangularly bent from
apical portion, gently narrowed apical 0.25 towards slender, subparallel apical portion;
right margin (dorsal view) distinctly roundly produced at about mid-length; prebasal
tooth short and blunt. Phallobase subsymmetrical in dorsal and ventral views. Distal
lobe generally very similar of that in H. saga and related species, overall more
stretched than compact; submembranous contorted distal portion relatively long and
wide; opening funnel-like and apicad directed (like in a tuba), located at the most right
(behind main piece and distal lobe trunk in dextrolateral view, Fig. 6B); most sclerotised
enlarged distal portion with conically-pointed apex in dorsal and ventral views (Fig. 6A).

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 13

Figure 6. EES

Hydraena dinarica, sp. n. aedeagus (holotype): A) dorsal view: B) lateral view; scale bar = 0.1
mm.

Female tergite X (Fig. 5E), except for suboval shape, very similar to those of H. saga
and related species (comp. Jach and Diaz (2017): Figs. 7 and 12); apex widely
rounded; disc with sub-basal squamose setae and with few trichoid setae; squamose
setae comparably elongate and not conspicuously widened apically; subapical fringe
admedially with dense fringe of vermiform setae of equal length which are slightly bent
in apical half and with few long trichoid setae laterally.

Gonocoxite (Fig. 5F) very similar to those of H. saga and related species (comp. Jach
and Diaz (2017): Figs. 6 and 11), subtrapezoidal; hyaline apex round; inner plate
moderately projecting basally and laterally; apical area densely pubescent; basal area
without setae; cavity oval.

Spermatheca not examined.

Secondary sexual characters: Female elytral apices produced and separately gently
rounded, not acuminate. All femora of male slightly more inflated. Male ventrite VI
enlarged (Fig. 5A). Male mesotibia with a row of ca. ten denticles along proximal half of
mesial face (Fig. 5G), in females only with setae (Fig. 5H). Male metatibia with fringe of
long setae at inner face of posterior half (Fig. 51), in females only with regular setae
(Fig. 5J).

Differential Diagnosis

Hydraena dinarica, sp. n. is morphologically very similar to species that are referred to
as H. saga complex (sensu Jach and Diaz (2017); see Discussion), namely H. alpicola,
H. diazi Trizzino, Jach & Ribera, 2011, H. emarginata Rey, 1885, H. fosterorum
Trizzino, Jach & Ribera, 2011, H. kahleni Jach & Diaz, 2017, H. larissae Jach & Diaz,

14

Freitag H et al

2000, H. saga and H. samnitica Fiori, 1904 (original descriptions by Fiori (1904), Jach
and Diaz (2000), Jach and Diaz (2017), Orchymont (1930a), Rey (1885), Trizzino et al.
(2011a)). All of them are most typically characterised by their articulate, contorted
aedeagal distal lobe and the rounded or truncate apex of the aedeagal main piece.
Hydraena belgica d’Orchymont, 1930; H. dalmatina Ganglbauer, 1901; H. hispanica
Ganglbauer, 1901; H. pangaei Jach, 1992; H. pelops Jach, 1995 and H. tarvisina
(Ferro, 1991) (original descriptions by Ferro (1991), Ganglbauer (1901), Jach (1992),
Jach (1995), Orchymont (1930b)) were additionally added by Trizzino et al. (2013a)and
defined as H. emarginata complex (which would also include H. /otti Bilton, 2013 (see
Bilton (2013)). They share the structural plan of the aedeagal distal lobe, but possess
an acute aedeagal main piece and lack the minute seta on the outer (right) side of the
main piece. Therefore, the latter species are not discussed here in detail, but it should
be noted that H. dalmatina might occur sympatrically and is externally quite similar, but
can be distinguished by the widely explanate elytral margin in apical third and its elytral
apices (acuminate in males, truncate in females).

In comparison with all species mentioned above, Hydraena dinarica, sp. n. is unique in
the tuba-like 180° bent hyaline distal tube of the aedeagus (Fig. 6) and thus upward
directed opening (vs. downward or lateral directed). Its distal portion is overall larger
than in all other species of the H. saga complex (as defined above), except for H.
emarginata, some specimens of H. /arissae and H. samnitica with subequally large
distal portion. Similarly, the aedeagus of H. dinarica, sp. n. is larger (main piece 630 um
long) than most species mentioned above (510-610 um), except for H. emarginata
(610-665 wm).

Within this complex, H. dinarica, sp. n. seems morphologically most similar to the
Italian species H. kahleni and H. larissae, especially based on their moderately large
contorted aedeagal distal lobe, as well as H. saga on the external habitus. While H.
dinarica, sp. n. is slightly larger (2.25-2.45 mm long) than the latter three species
(1.95-2.30 mm long), the elytral disc appears slightly flatter and the elytral margin very
slightly more explanate in H. dinarica, sp. n. The elytral apices are similar and within
the observed variation range in the former species in both sexes. The new species also
resembles H. samnitica of almost the same size (especially in the moderately large
contorted aedeagal distal lobe), but it is externally distinguishable from H. samnitica by
the explanate elytral margin extending almost up to the apex (vs. reaching apical 0.15;
the apical area therefore appears more slender in H. dinarica, sp. n.).

On the other hand, H. dinarica, sp. n. seems genetically closest to H. alpicola, H. saga
and the H. gracilis Germar, 1824 complex (as defined by Jach (1995)), based on the
DNA barcode (Fig. 7). H. saga occurs in the region (material at NMW/; the closest
known collection site is in Foca, Bosnia, less than 50 km away from the type locality of
H. dinarica, sp. n.) and is also most similar. Therefore, the species can only reliably be
identified by dissection of its aedeagus.

Males can be distinguished as stated above, while in females of H. dinarica, sp. n., the
gonocoxite (Fig. 5F) is subtrapezoidal, with evenly round and expanded apical hyaline

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 15

area (vs. subquadrate gonocoxite and with short apical area in H. saga; Jach and Diaz
(2017): Fig. 11). Furthermore, the female tergite X in H. dinarica, sp. n. (Fig. 5E) is
suboval, its basal portion expanded and the vermiform setae of the subapical fringe are
bent (vs. subtriangular tergit X with short basal portion and with almost straight
subapical vermiform setae in H. saga; Jach and Diaz (2017): Fig. 12).

@ Montenegro p- § H68 BC12 @ H. dinarica sp.n.
@ Austria
@ Bulgaria

fe)

@ Finland
@ France

@ Germany

@ Greece
@ Italy
@ Spain

H. britteni @——co——®
kJ964025

H. sp. female
H. biltoni cs He6 BC11 @

Q

H. morio
H62 BCO8
H64 BCO9
H70 Sanger
H71 Sanger

66

H. minutissima
KM443241

H. minutissima aT
5

H. melas @& {27}

H. britteni @ ku919146

KU917753

H60 BCO7

KU909065
KU916026

KUO68 77, H. saga

HesTpO0e H. alpicola

H. gracilis
LT855685

H. gracilis @
KM444579
KU907634
KU909048
KU909473
KU909933
KU915350
KU919346
LT855682

¢
f°) LT855684

H. cf. britteni

H65 BC10

KU907756
—® HH. excisa eo 9
KU916374
27

H. gracilis

@ H. gracilis
KU912591

H. gracilis
HM909038

HF947945
H. fosterorum
HF970922

PSS ele H. larissae

HE970890

HR97089:
4

HE970906

H. nigrita @—C

ets {9} T {12+~e
30 38

9
tT H. subintegra

3
aval H. croatica 63 oth H. subintegra

KU917836

0}
H. nigrita he BCO08

Figure 7. EES

Statistical parsimony haplotype network of successfully sequenced samples of Hydraena
dinarica sp. n., H. biltoni, H. minutissima, H. morio, H. nigrita, H. subintegra, two unidentified
female specimens and GenBank records of related species from aligned CO/ sequences of
567 bp length.

Externally, the new species also resembles other representatives of the "Haenydra"
lineage which might occur sympatrically, but differ in certain characters: Hydraena
bosnica Apfelbeck, 1909 (posteriorly sinuate pronotum, oval elytra; Trizzino et al.
(2013a): Fig. 42a); H. excisa Kiesenwetter, 1849 (clypeus shagrened; elytra less
elongate, more oval with disc more convex, margin more explanate, apices slightly
truncate in males, sharply (not roundly) notched in females; Trizzino et al. (2013a): Fig.
32l); H. gracilis balcanica d'Orchymont, 1930 (elytra with disc more convex, margin
less explanate, apices almost conjointly rounded in males; Trizzino et al. (2013a): Fig.
24a); H. phallica d'Orchymont , 1930 (clypeus shagrened; elytra more oval with disc
more convex, apices slightly truncate in males, shallowly notched in females; 7rizzino
et al. (2013a): Fig. 32p); H. subintegra (smaller and paler, elytra reddish-brown with
disc more convex, margin more explanate, apices conjointly rounded in both sexes); H.
vedrasi d’Orchymont, 1931 (elytra with disc more convex, apex subtruncate in males,
sharply (not roundly) notched in females; Trizzino et al. (2013a): Figs. 22a and g)

16

Freitag H et al

(original descriptions by Apfelbeck (1909), Kiesenwetter (1849), Orchymont (1930a),
Orchymont (1930b), Orchymont (1931)).

Hydraena dinarica, sp. n. varies by 0.6% genetic distance (657 bp CO7 barcode from
the most similar congeners H. alpicola and H. saga and by 3.3% from the
morphologically similar H. /arissae (Suppl. material 1).

Habitat

This species was collected from a mountain stream in a forested, undisturbed karst
area at an altitude of about 1220 m a.s.l. (Fig. 2). During the time of collection, the river
was only partly on the surface; the predominant flow was subsurface, causing low
water temperatures. The well-shaded riverbed, including the water-bearing reaches,
was densely covered with mosses. The specimens were collected from the upper
interstitial of the bottom gravel (nesopsammon) in shallow, partly rapidly flowing water.

Distribution

So far only known from the type locality at the northern slopes of Durmitor Mt.,
Montenegro (Fig. 8).

Figure 8. EES]

Map of Europe with the collection site of Hydraena dinarica sp. n. and the distribution of
morphologically similar species of the "Haenydra" lineage defined as "Hydraena emarginata
complex" by Trizzino et al. (2013a) and H. gracilis as genetically similar species with
overlapping range.

Etymology

The species is named after the Dinaric Alps, or Dinarides, a karst mountain range
where Durmitor Mt. and the type locality of the new species are situated. The epithet is
used as an adjective meaning "of the Dinaric Alps".

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 17

Analysis
DNA barcoding

The amplification of all sequences but one (H67), with LCO1490 & HC02198 primers and
the applied protocols, was successful (Table 1).

Table 1.

GenBank accession numbers of Hydraena CO/ mtDNA barcode sequences generated in this study.

Species Specimen Site Voucher Sequencing ENA BOLD

H. dinarica sp. n. female MNE18c H68 Run1 BC12 MT784148 TXEX049-20
H. dinarica sp. n. male MNE18c H67 - - -

H. subintegra male MNE17c H63 Runi BCO9 MT784149 TXEX051-20
H. nigrita male MNE17b H61 Runi BCO8 MT784150 TXEX052-20
H. sp. (cf. britteni) female MNE101I H65 Runi BC10 M1T784151 TXEX053-20
H. sp. female MNE13k H66 Runi BC11 M1T784152 TXEX054-20
H. morio male MNE17b H62 Run2 BCO08 M1T784154 TXEX055-20
H. morio female MNE17b H64 Run2 BCO9 MT784155 TXEX056-20
H. morio male MNE17b H71 Sanger MT784156 TXEX057-20
H. morio male MNE17b H70 Sanger MT784157 TXEX058-20
H. biltoni male MNE21c H69 Sanger MT784158 TXEX050-20
H. minutissima male MNE17b H60 Run1 BCO7 MT784159 TXEX059-20

One repository sequence (KU906877), labelled "Hydraena gracilis", does not cluster
together with the remaining samples of this species, but rather with H. a/pico/a Pretner,
1931 (a species found around the northern and eastern Alps and first recognised by
Pretner (1931)), to which its barcode is identical. Given that: (1) the underlying study is a
multi-taxon methodological paper testing a bioinformatic pipeline and not a taxonomic
contribution (Rulik et al. 2017); (2) species of the H. gracilis main clade are often hardly
distinguishable without dissection of the male genitalia; (3) if not exactly the same place,
the collection site of the sample is nonetheless in close proximity to the type locality of H.
saga d'Orchymont, 1930(b), while there is no published record of a real H. gracilis from any
nearby locality; (4) H. alpicola and H. saga are known to have sometimes identical CO/
sequences (Trizzino et al. 2011a); (5) the margin of the distribution area of H. alpicola is
more than 300 km away from that collection site (Trizzino et al. 2013a), we assume with
reasonable confidence that the said barcode belongs to a H. saga specimen and treat it as
such herein.

18 Freitag H et al

Interspecific genetic distances ranged from 0.0-17.8% (Suppl. material 1). While Hydraena
alpicola and H. saga can obviously not be distinguished by their barcode, the highest
divergence was observed for H. subintegra and H. minutissima as representatives of
different main lineages of the same subgenus, Hydraena (s.str.).

Discussion

Due to the recent speciation, it is not surprising that we can barely delineate the species
complex nor the individual new species by employing a 2% or 3% species delineation
threshold of the mtDNA barcode as originally proposed (Hebert et al. 2003). This aspect is
worth noting as it will be impossible to differentiate many young Hydraena species by the
classical use of DNA barcodes.

Mitochondrial genes, in general and thus standard barcodes, often lag behind in terms of
lineage sorting compared to nDNA involved in speciation (e.g. Monaghan et al. 2006, Balke
et al. 2013) and this mitochondrial genetic signal can be too unstructured amongst recently
radiating species (Meyer and Paulay 2005, Hendrich et al. 2010).

The habitat of Hydraena dinarica is quite typical for some highly specialized
representatives of the "Haenydra" lineage. A similar habitat was also figured for the
recently discovered Hydraena kucinici Micetic Stankovic & Jach in the Republic of
Macedonia (Miceti¢ Stankovic and Jach 2012: Figs. 1 and 2). The post-glacial, cold water
habitat of H. dinarica suggests that it has evolved from a relictual population of a former
cold-temperate/sub-polar climate-adapted ancestor related to H. saga, which had moved
its distribution area southwards during the Pleistocene. Most probably, the new species
could not survive in the lowlands of current Montenegro or even in regular surface waters
of the same altitude, as reflected by its very restricted habitat and distribution. It can be
assumed that the species withstands periods without surface flow (e.g. late summer)
hidden in the hyporheic zone.

Hydraena dinarica clearly belongs to the "Haenydra" lineage (formerly regarded as
subgenus by Berthelemy (1986) and Perkins (1997), a clade that was estimated to have
occurred in the late Miocene (Tortonian), but however, is not satisfactorily resolved in terms
of its phylogenetic position within the genus up to now (Trizzino et al. 2013b). Within the "
Haenydra" lineage, H. dinarica, sp. n. belongs to the H. gracilis main clade of about 7-8
Ma age, which is morphologically evident in the articulated aedeagal distal lobe, as well as
the low genetic distance from other representatives, based on the 657 bp barcode (Suppl.
material 1). The very high morphological similarity, particularly in the articulate aedeagal
distal lobe, gonocoxite and female tergite X, as well as the mtDNA data, clearly identify the
new species as closely related to H. saga and H. alpicola. The terminology for the group, to
which the latter two and other close congeners belong, is rather confusing and
inconsistent. Most recently, it is referred to as H. saga complex sensu Jach and Diaz
(2017), previously as the H. emarginata / H. emarginata-saga complex in a wider sense
(Trizzino et al. 2011a, Trizzino et al. 2013a) including the H. belgica complex sensu Jach
and Diaz (2017), but all of them are not identical to the “HW. saga complex” as mentioned by

Hydraena (s.str.) dinarica, new species (Coleoptera: Hydraenidae) along ... 19

Ribera et al. (2011), referring only to the Iberian Peninsula representatives H. diazi and H.
fosterorum. All of the latter are not well supported by DNA data, where H. saga and H.
alpicola (of which mtDNA haplotypes have been reported to be sometimes identical
(Trizzino et al. 2011b) usually cluster as a sister sub-clade to the H. graciles sub-clade,
rather than with the other members of this group (current study, Ribera et al. 2011, Trizzino
et al. 2013a). Irrespective of their morphological similarity, the polyphyletic appearance of
the eastern (H. alpicola, H. dinarica, H. saga and possibly H. kahleni for which no genetic
data are published) and western (all remaining species of the H. saga complex sensu Jach
and Diaz (2017)) sub-complexes are not congruent with either definition of such species
complex. Redefining the species complexes appears necessary when more genetic
information, including nuclear markers, becomes available.

Based on the DNA data available (e.g. current study, Abellan and Ribera 2017, Ribera et
al. 2011, Trizzino et al. 2011b), it seems most reasonable to consider the eastern
representatives and presumed sister group of the H. gracilis complex (Jach 1995) as a
newly-defined H. saga complex which, however, is not the purpose of this paper.

Many of these species are known to be highly endemic and all of them are young species,
some of the closest relatives, such as H. saga and H. alpicola, have just split recently
during the Pleistocene (about 50,000 years ago, based on data of Trizzino et al. (2013b)).
A comparably young age can also be assumed for H. dinarica, sp. n. which coincides with
the Wurm glaciation that covered large parts of Durmitor massif in ice for the last time
(Djurovi¢ 2009). Ribera et al. (2011) demonstrated a strong non-randomness of the
geographic distribution of the species in some "Haenydra" clades, meaning phylogenetic
distance is congruent to geographic distance of the related species (Fig. 8). This seems to
be evident also for H. dinarica, sp. n. of which H. saga is the geographically closest
member of the young species complex and is probably also the closest genetically.
Therefore, post-glacial range fragmentation, due to the developing Mediterranean climate
with a pronounced arid season, appears to be the most likely scenario for the evolution of
H. dinarica, sp. n. The obvious ecological preference (or restriction) to its regionally-rare
microhabitat with a partly subterranean flow of cold water supports this assumption.

Its rarity, its presumably very limited distribution range and its special habitat association
suggest that the new species is particularly vulnerable to climate change and habitat
destruction.

Acknowledgements

Specimens were collected under Permit 02-UPI-662/4 kindly issued by the Environment
Protection Agency of Montenegro. The first author wants to thank the curators Dr. Manfred
A. Jach (Natural History Museum Vienna) and Olaf Jager (Senckenberg Natural History
Collections Dresden) for access to and loan of their collections. The whole team would like
to thank the staff of Etno selo Slieme for their hospitality and for allowing us to set up a
laboratory in a part of their restaurant.

20 Freitag H et al

Author contributions

Conceptualisation, HF, MD & IN; collection and identification of material, HF, RdV, CGT,
HL, RL, MFF, MG & CVP; formal species description, HF & RdV; MinlION sequencing, MP,
SM, MD; preparation of Sanger sequencing, HF, ED, MRS; molecular data analysis, ED &
MRS; writing—original draft preparation, HF, RdV, CGT, MP, SM, IN; writing—review and
editing, all authors.

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Supplementary materials

Suppl. material 1: Intra- and interspecific CO/ sequence divergence (K2P). EE}

Authors: Hendrik Freitag

Data type: genetic divergence table

Brief description: Specimens sequenced in the field by the use of the ONTrack MinlON pipeline
are indicated by their "H[number]" code. Sequences of Hendrich et al. (2015), Pentinsaari et al.
(2014), Ribera (2011), Rulik et al. (2017), Trizzino et al. (2011b; 2013b), are indicated by their
respective GenBank accession numbers.

Download file (12.27 kb)

Suppl. material 2: Taxon Expedition: the exciting discovery from Durmitor Mountain

Authors: Clister V. Pangantihon
Data type: multimedia
Download file (5.85 MB)