The genus Hyphydrus Illiger, 1802 represents a well-defined group of medium sized, globular shaped Dytiscidae. Altogether 139 species occur in all regions of the Old World, with most species distributed in tropical Africa (Miller and Bergsten 2016; Nilsson and Hájek 2017a). A taxonomic revision of the genus was published by Biström (1982).

Only three Hyphydrus species occur in Europe (cf. Nilsson and Hájek 2017b). While the Mediterranean H. aubei Ganglbauer, 1891 can be easily identified based on black markings on ferrugineous dorsal surface, the uniformly dark-ferrugineously coloured H. anatolicus Guignot, 1957 is very similar to the widespread western Palaearctic H. ovatus (Linnaeus, 1760) and it was not recognised until 1957. Hyphydrus anatolicus was described originally from Angora [= Ankara], Turkey (Guignot 1957). Subsequently Sanfilippo (1963) described the same species under the name H. carrarai Sanfilippo, 1963 from Italy. The synonymy of both species was established by Pederzani (1976). The species was later included in the revision of Biström (1982), who synonymized H. anatolicus with the older name H. sanctus Sharp, 1882, known previously only from the Levant region. Biström (1982) also argued that H. sanctus and H. ovatus should possibly be regarded as subspecies, but that more work was needed. Although Wewalka (1984) described the differences between H. anatolicus and H. sanctus, and a habitus photo of H. anatolicus was published by Hájek (2009), both mentioned species remain enigmatic, predominantly because of their similarity with H. ovatus, and because their distribution is not satisfactorily known.

The extraction protocol was different for the fresh alcohol-material of H. ovatus and H. anatolicus (from Russia) versus the dry-mounted older material of H. anatolicus and H. sanctus. The former was extracted in 96-well Wizard SV plates following the manufacturers instructions (Promega). The 3’ end of cytochrome c oxidase subunit 1 (CO1) was amplified with the primers PatDyt or RonDyt (Isambert et al. 2011) and Jerry (Simon et al. 1994) using 1ul of DNA, Bioline Taq and the following cycling conditions: 94° for 2min, 35 to 40 cycles of 94° for 30s, 51–53° for 60s and 70° for 90-120s, and a final extension of 70° for 10 min. PCR products were cleaned with a 96-well Millipore multiscreen plate, sequenced in both directions using a Big Dye 2.1 terminator reaction, and analysed on an ABI 3730 automated sequencer. PatDyt and Jerry were used as sequencing primers. The older dry-mounted specimens were extracted using the QIAamp® DNA Micro Kit (QIAGEN®), following the tissue protocol with the addition of 20ul of DTT (Dithiothreitol)(Sigma-Aldrich). PCR was done with a set of 6 newly designed primer pairs (Table 2) amplifying the complete 825bp CO1 segment in shorter overlapping segments between 147 and 228bp long. We used Ready-ToGo™ PCR beads (Amersham Biosciences) together with 1ul of 10uM of each primer, 2ul of DNA and 21ul water in a 25ul reaction. Cycling conditions started with a 5 min denaturation step at 95°C followed by two cycles of 30 s at 95°C, 30 s at 45°C (first, second and fourth fragments) or 50°C (third, fifth and sixth fragments), and 40 s at 72°C, then two cycles of 30 s at 95°C, 30 s at 43°C or 48°C and 40 s at 72°C, and 39 cycles of 40 s at 95°C, 40 s at 41°C or 46°C, 50 s at 72°C, then a final extension step of 8 min at 72°C. PCR reactions were purified with Exonuclease I and FastAP (Fermentas) in the proportion 1:4, and sequenced with a BigDye™ Terminator ver. 1.1 Cycle Sequencing Kit (Applied Biosystems), cleaned with a DyeEx 96 kit (QIAGEN) and run on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequences are submitted to Genbank under accession codes FN998871-FN998899 and JX221701- JX221703.

Sequences were assembled and edited in Sequencher 4.8 (Gene Codes Corporation) and aligned in ClustalX 2.0 (Larkin et al. 2007) with default settings of 15 as gap opening penalty and 6.66 as gap extension penalty. The alignment contained no gaps. Bayesian analysis was done with MrBayes 3.2.1 (Ronquist et al. 2012). We set up a partitioned model based on 3^rd resp. 1^st+2^nd codon positions and applied a HKY+G+I model to each partitions, unlinking statefrequencies, t-ratio, shape and proportion of invariable sites. Partitions were allowed separate rates with a variable rate prior. All other prior and proposal settings were left as default. We ran two separate runs each with four chains (one cold and three incrementally heated) 3 million generations sampled every 1000^th generation. First 25% was discarded as burn-in. For the first analysis we used a time-free model and rooted the tree with the outgroup Hyphydrus aubei. For the second analysis we excluded the outgroup and instead tested the placement of the root with a clockmodel. We used a Bayes Factor test to assess if the data was compatible with a strict molecular clock or if a relaxed clock should be used. A heuristic parsimony analysis was run in Nona (Goloboff 1999) (hold 10000, Mult*100, hold/10, mult*max*) spawned from Winclada (Nixon 1999-2002). The parsimony analysis was followed by optimising the characters on the most parsimonious tree. This was done to show discrete character support for the three species. We performed a species-delimitation analysis using the general mixed yule coalescence model (GMYC) as implemented in R (R Development Core team 2005) with the package Splits (Ezard et al. 2009; Fujisawa and Barraclough 2013). We tested the null-hypothesis that the Hyphydrus ovatus-complex is a single species versus the alternative hypothesis that it consists of more than one species with a likelihood ratio test under the GMYC model. The GMYC method optimizes the likelihood of a single threshold across an ultrametric gene-tree. The threshold defines speciation branches towards the root from the threshold and within-species coalescence branches towards the tips from the threshold. The older branches are modelled with a Yule (speciation) model while the younger branches are delimited into n-groups where each group is modelled with a separate coalescent process model. The maximum likelihood solution of the GMYC model (the likelihood is calculated placing the threshold at each node across the tree) is compared against a model treating the entire gene-tree as a single coalescence (i.e. as a single species) in the likelihood ratio test. We used the ultrametric clock-tree generated above as input to the species delimitation test.

The specimens were examined using an Olympus SZX12 stereomicroscope. Measurements were taken with an ocular graticule. Habitus photographs were taken using a Canon MP-E 65mm f/2.8 macro lens with 5:1 optical magnification on bellows attached to a Canon EOS 550D camera. Drawings were made based on photographs taken using an Olympus SZX12 microscope equipped with a Canon EOS 1100D digital camera. Images of the same specimen/structure at different focal planes were combined using Helicon Focus 5.1.19 software. To avoid artefacts due to desiccation of poorly sclerotised parts, the genitalia were illustrated mounted in dimethyl hydantoin formaldehyde resin (DMHF) on the same card as the beetle.

Amplification was highly successful with the short fragment PCRs of old dry-mounted material (Table 3). The full-length 825bp segment was achieved for the two H. sanctus specimens from Israel, 665bp for one of the Turkish specimens, and a 147bp segment of the second Turkish specimen, with three ambiguous base calls. The last specimen also gave a 175bp sequence from primer pair 1 (Table 2) that turned out to be contaminated DNA with closest BLAST hit on Genbank being saccharomycete fungi. This is always a risk when extracting DNA from the whole body of a specimen. All three dry-mounted H. anatolicus specimens yielded full-length CO1 sequences.

Genetic distances between the three presumed species in the ovatus-complex turned out to be large (Table 4). The distance between H. ovatus and H. anatolicus or H. sanctus was 9.4–11.4% (K2P-model). The distance between H. sanctus and H. anatolicus was slightly less, 6.7–7.1%. These genetic distances strongly indicate that we are dealing with three valid and separate species in the ovatus-complex. Within-species variation was less than 1.4%. The time-free bayesian analysis as well as the parsimony analysis, both rooted with H. aubei as outgroup, confirmed that the three presumed species are reciprocally monophyletic and separated from each other with long branches (Figs 1–2). Posterior probability support values were 1.0–0.98 for all three species. H. sanctus and H. anatolicus are sister species according to this single-gene phylogeny both in the outgroup-rooted trees (Figs 1–2), and in the clock-rooted tree (Fig. 3). Parsimony analysis and character optimization confirmed the H. sanctus + H. anatolicus sister group relationship with 17 supporting unambiguous and non-homoplasious substitutions (Fig. 2). Also all three presumed species were supported with between 16 and 24 unambiguous and non-homoplasious substitutions (Fig. 2). The Bayes factor test strongly favoured the strict clock (LnL=-1701) over a time-free model (LnL=-1764) (2*LnBF=125), hence a strict, as oppose to a relaxed, clock model was used to generate an ultrametric tree (Fig. 3). The GMYC model delimited three clusters congruent with the three presumed species as the maximum likelihood solution (Fig. 3). An approximate confidence interval of 2log likelihood units from the maximum likelihood (3 clusters) did not include any other solution. The explicit likelihood ratio test of the null hypothesis of a single coalescing unit (species) was refuted in favour of the alternative hypothesis of three separately evolving and coalescing units (-Log L_{one species}= 211.9965, -Log L_{three species}= 218.2261, Likelihood ratio=12.4592, p=0.00596).

Figure 1. 

Majority-rule consensus tree from the non-clock Bayesian analysis. Posterior probability clade support values >0.9 shown. Country abbreviations: SW=Sweden, GE=Germany, UK=United Kingdom, La=Latvia, RU=Russia, TU=Turkey, IS=Israel, GR=Greece, SL=Slovakia. Rooted (midpoint) with Hyphydrus aubei.

Figure 2. 

One of 14 most parsimonious trees (L=203, zero-length branches hard-collapsed) with unambiguous characters optimized. Black dots=non-homoplasious characters, white dots=homoplasious characters. Numbers refer to the character’s position in the alignment from 1-825. The other 13 cladograms only differed in within-species internal organizations. Rooted with Hyphydrus aubei. Country abbreviations as in Figure 1.

Figure 3. 

Clock-rooted ultrametric tree from Bayesian analysis with branches coloured according to the GMYC species delimitation analysis. Posterior probability clade support values >0.9 shown. Black branches=speciation events, red braches=within species coalescence events. Country abbreviations as in Figure 1.

All mentioned species belong to the Hyphydrus ovatus species group sensu Biström (1982). The group contains nine species occurring exclusively in the Palaearctic region. The members of the group are well characterised with the longer metatibial spur of males serrate (cf. Fig. 7). The four western Palaearctic species share the similar shape of the median lobe of aedeagus which is rather poorly sclerotised, in ventral view nearly parallel-sided with sides straight, very slightly and continually narrowing from base to apex (cf. Fig. 8). Finally, the three species of the H. ovatus complex (i.e. H. anatolicus, H. ovatus and H. sanctus) can be easily recognised by their more or less uniform dark ferrugineous to ferrugineous body colouration, rarely with minor pale markings.

Due to rather weak sclerotisation of external genitalia, the genital characters have only limited use for identification of species in this complex. Therefore, we focused more on habitus, punctuation and structure characters of the species. The most diagnostic character is probably the shape of the longer metatibial spur on males (see Fig. 7). A key to identification of all western Palaearctic species of the H. ovatus species group is presented at the end of the taxonomic section.

Hyphydrus anatolicus Guignot, 1957

Hyphydrus anatolicus Guignot, 1957: 91 (orig. descr.; type locality: “Angora” [Ankara, Turkey]).

Hyphydrus carrarai Sanfilippo, 1963: 77 (orig. descr.; type locality: “Macchia di Migliarino, Torre del Lago (Toscana)” [Italy]); synonymy by Pederzani 1976: 166.

Hyphydrus sanctus : Biström 1982: 39 (partim, misidentification).

Published records

Bosnia and Hercegovina: Biström (1982: 39 as H. sanctus). Croatia: Guéorguiev (1971: 8 as H. carrarai); Biström (1982: 39 as H. sanctus); Ádám (1992: 194 as Hyphydrus sanctus); Temunović et al. (2007: 17); Krčmar (2014: 20). Greece: Biström (1982: 39 as H. sanctus); Wewalka (1984: 131). Hungary: Ádám (1992: 194 as H. sanctus); Csabai et al. (1999: 148 as H. sanctus); Móra et al. (2004: 153); Csabai and Nosek (2006: 73); Kálmán et al. (2008: 76); Molnár (2008:110); Sóos et al. (2008: 223); Lőkkös (2010:161). Italy: Sanfilippo (1963: 77 as H. carrarai); Angelini (1972: 182 as H. carrarai; 1984: 54); Pederzani (1976: 166); Biström (1982: 39 as H. sanctus); Rocchi (1991: 68); Pederzani & Campadelli (1996: 21); Nardi (1997: 132); Bordoni et al. (2006: 87). Macedonia: Biström (1982: 39 as H. sanctus). Montenegro: Scheers (2016: 209). Russia: Biström (1982: 39 as H. sanctus). Serbia: Mesaroš (2015: 50). Turkey: Guignot (1957: 91).

Material examined

Greece: 2♂♂, Ionian Islands, Kerkyra, Chalikiopoulos [lagoon], 22.iv.1935 (NHMW); 1♂, Eastern Macedonia and Thrace, Évros Distr., plain of Évros river, 26.vii.1988, M. Jäch leg. (NHMW); 1♀, Central Macedonia, Khalkidhiki Distr., Sithonia, 2 km S of Kalamítsion, 12.viii.2000, J. Hotový leg. (NMPC); 5♂♂ 5♀♀, NW Peloponnese, 3 km S Kalogria, 38.1213N, 21.3810E, ca. 3 m, shallow seasonal swamp, 17.v.2010, H. Fery & L. Hendrich leg. (NMPC). Hungary: 1♂ 1♀, Hungary 46 36 (BMNH); 1♀, Bács-Kiskun, Kiskunmajsa env., 11.viii.1999, J. Hájek leg. (NMPC). Montenegro: 1♂, Vranjina env., Skadarsko jezero, 20.ix.2001, J. Hájek leg. (NMPC). Russia: 1♀, Orenburg reg., Totskoye, 1917, Š. Jureček leg. (NMPC); 2♂♂ 2♀♀, Samara, K. Fausta leg. (ZMAS); 1♀, Stavropol reg., Kuma river, 20.iv.1911 (ZMAS); 1♂ 2♀♀, Volgograd reg., 2 km south of Zryanin village, 48°36‘60‘‘N, 43°10‘10‘‘E, small lakes near Liska river, incl silty open bay with grasses, Alisma and Juncus, 29-30.iv.2002, J. Bergsten & A. Nilsson leg. (NHRS); 1♂ 1♀, Volgograd reg., Archeda-Don rivers alluvial sandy plain, 16 km ESE of Terkin village, 49.6861N, 43.3333E, different lakes, grassy ponds, fens and stream, 2-3.v.2002, J. Bergsten & A. Nilsson leg. (NHRS); 3♂♂ 2♀♀, Volgograd reg., Kretskiy, 48.6083N, 44.7061E, river-arm, newly flooded grassland, 5.v.2002, J. Bergsten & A. Nilsson leg. (NHRS). Turkey: 2 spec., Aydin vil. [= province], S of Aydin, ditch, 4.iv.14987, H. Fery leg. (HFCB); 4♂♂ 1♀, Muğla vil. [= province], Köyçeğiz, 27.v.1991, S. Schödl leg. (NHMW, NMPC). Slovakia: 1♂, 1 km N of Tvrdošovce, 24.iv.2000, T. Kopecký leg. (NMPC). Ukraine: 1♂, Kherson distr., monast. Korsunskij, cursus inf. fl. Dnjepr, 3.vi.1927, S. Medvedev leg. (ex coll. Zakharenko, ZMAS).

Diagnosis

Habitus as depicted in Figs 4c, 5c. Clypeus with anterior margin rounded (Fig. 6a). Reticulation of dorsal surface confined to head, more distinct and impressed anteriorly. Punctation of head fine, visible on whole surface; punctures sparse, distance between them usually equal or bigger than their diameter (Fig. 6a). Punctation of pronotum double, fine, distance between larger punctures bigger than their diameter. Punctation of elytra double, diameter of small puncture less than half of diameter of large punctures; distance between large punctures bigger than their diameter. Epipleura smooth with fine punctures. Metatibia with sinuous outer margin.

Figure 4. 

Hyphydrus male habitus. a H. aubei (Corsica; 4.9 mm) b H. ovatus (Sweden; 5.0 mm) c H. anatolicus (Slovakia, specimen post-extraction; 5.1 mm) d H. sanctus (Turkey; 5.2 mm).

Figure 5. 

Hyphydrus female habitus. a H. aubei (Croatia; 4.7 mm) b H. ovatus (Bohemia; 4.6 mm) c H. anatolicus (Greece; 5.0 mm) d H. sanctus (Turkey; 4.9 mm).

Figure 6. 

Hyphydrus head. a H. anatolicus b H. ovatus c H. sanctus. Not in scale.

Male. Longer metatibial spur long, nearly as long as metatarsomere I-II combined (Fig. 7a); spur bisinuate with only indistinct serration basally (Fig. 7a). Male genitalia as in Fig. 8a–d, median lobe in ventral view slightly narrowing from base to apex.

Female. Both shiny and matt forms known of females of H. anatolicus. Shiny form agreeing well with male; matt form with whole surface densely reticulated, meshes somewhat elongate on elytra. Large punctures well visible, small punctures indistinct among reticulation. Longer tibial spur shorter than in male; broad and with serration in basal two thirds, narrowed, slightly curved and without serration in apical third. Female genitalia as in Fig. 8e–g.

Habitat

The species inhabits various types of standing water, predominantly densely vegetated pools, ditches and small ponds. H. anatolicus tolerates also saline habitats.

Distribution

The species is distributed in the Eastern Mediterranean and in south-eastern Europe. It occurs in Italy, southernmost Slovakia, Hungary, the Balkan Peninsula, Turkey, southern Ukraine and Russia up to latitude 55° and east to the Ural Mountains (Fig. 9). First record from Slovakia and Ukraine.

Figure 7. 

Hyphydrus male metatibia, longer metatibial spur and metatarsomere I. a H. anatolicus b H. ovatus c H. sanctus. Scale bar 0.5 mm.

Figure 8. 

Hyphydrus male and female genitalia. a, h, o median lobe of aedeagus in ventral view b, i, p supplementary drawing of apex of median lobe c, j, q median lobe of aedeagus in lateral view d, k, r paramere e, l, s gonocoxa f, m, t gonocoxosternite g, n, u spermatheca. a–g H. anatolicus h–n H. ovatus o–u H. sanctus. Scale bar 0.5 mm.

Figure 9. 

Map of distribution of H. anatolicus (circles, dots) and H. sanctus (squares). White symbols represent records from the literature, large circles represent imprecise data for a larger region (country); black symbols represent records of specimens examined by us.

Key to western Palearctic species of the Hyphydrus ovatus species group