Iberoporus pluto sp. n., the first stygobiont beetle from Portugal (Dytiscidae, Hydroporinae), is described from a single female from the cave Soprador do Carvalho (Coimbra). The species is highly troglomorphic, depigmented, blind, and with elongated appendages not adapted for swimming. A molecular phylogeny based on a combination of three mitochondrial and two nuclear genes showed the new species to be sister to I. cermenius Castro & Delgado, 2001 from Córdoba (south of Spain), within the subtribe Siettitiina of the tribe Hydroporini. Both species are included in a clade with Siettitia avenionensis Guignot, 1925 (south of France) and Rhithrodytes agnus Foster, 1992 and R. argaensis Fery & Bilton, 1996 (north of Portugal), in turn sister to the rest of species of genus Rhithrodytes Bameul, 1989, in what is here considered the Siettitia group of genera. We resolve the paraphyly of Rhithrodytes by transferring the two Portuguese species to Iberoporus Castro & Delgado, 2001, I. agnus (Foster, 1992), comb. n. and I. argaensis (Fery & Bilton, 1996), comb. n.

Diving beetles, groundwater, new species, stygofauna, troglomorphy

The knowledge of the subterranean fauna from Portugal has significantly increased over the last decade, with the description of a high number of obligate subterranean species (tripling their number) and the establishment of new biogeographic patterns (Reboleira 2012). A high number of these species are stygobiont (i.e., confined to groundwater), mostly from wells in the north of the country, where evapotranspiration is higher (Reboleira et al. 2011, 2013). They include 62 species of crustaceans, mostly asellids, syncarids and amphipods, and one species of annelid (Reboleira et al. 2013).

In this work we describe the first stygobiont species of Coleoptera from Portugal, a diving beetle of the subtribe Siettitiina (Dytiscidae, Hydroporinae, Hydroporini; type genus: Siettitia Abeille de Perrin, 1904). Siettitiina includes the only known European genera of Dytiscidae which have stygobiont members: Siettitia, with two species in France, Iberoporus Castro & Delgado, 2001, with one species in south Spain, Etruscodytes Mazza et al., 2013, with one Italian species, and Graptodytes Seidlitz, 1887, with the Moroccan G. eremitus Ribera & Faille, 2010 among several epigean members (Ribera and Faille 2010, Nilsson and Hájek 2018a). The subtribe also includes some North American stygobiont species, with an uncertain phylogenetic position (Miller et al. 2013, Kanda et al. 2016, Miller and Bergsten 2016, Nilsson and Hájek 2018b). The new species is known from a single female found in a well-studied cave in central Portugal. Despite multiple visits to the same cave no additional specimens have been found, so we describe here the species on the basis of its morphological singularity and of the molecular data that places it unambiguously among the west Mediterranean species of Siettitiina.

The two BEAST analyses (GTR and HKY evolutionary models) resulted in identical topologies and very similar branch lengths, although convergence for GTR evolutionary models was poor for some genes (nad1, 18S), so we present here only the results of the HKY models (Fig. 1). The topology was also almost identical to that obtained with RAxML (Fig. 1).

Figure 1. 

Phylogeny of the Siettitia group of genera, obtained with Bayesian methods. Numbers in nodes, Bayesian posterior probabilities/maximum likelihood bootstrap support (obtained in RAxML); c.n., constrained node in the Bayesian analysis. See Table 1 for details on the specimens.

We obtained a well-supported, well-resolved phylogeny of Siettitiina (Fig. 1). In agreement with previous results we recovered three clades, Graptodytes+Metaporus, Stictonectes Brinck, 1943 + Porhydrus Guignot, 1945, and the Siettitia group of genera as here defined, including Siettitia, Rhithrodytes, and Iberoporus (plus most likely Etruscodytes, see Discussion). The new species was placed as sister to Iberoporus cermenius Castro & Delgado, 2001 with strong bootstrap support (BS = 73%), although in the Bayesian analyses the support was lower (posterior probability, pp = 0.73). Both species were in turn sister to Rhithrodytes argaensis Bilton & Fery, 1996 plus R. agnus Foster, 1992 in a very well supported clade (BS = 94; pp = 0.97), and then to Siettitia (Fig. 1). All other sampled species of Rhithrodytes were placed as sister to this clade, rendering the genus paraphyletic. In order to preserve the monophyly of Rhithrodytes we thus transfer the two species to the genus Iberoporus, Iberoporus agnus (Foster, 1992) comb. n. and Iberoporus argaensis (Bilton & Fery, 1996), comb. n.

According to our calibration, the separation between the new species and Iberoporus cermenius was dated at ca. 10 Ma (95% HPD 13.4-6.9 Ma), with a similar age for the split from I. agnus + I. argaensis (11.4 Ma [15.0-8.3]), during the Tortonian (Fig. 1).

We obtained for the first time a phylogeny of Siettitiina including a species of its type genus, Siettitia. Despite the incomplete data, there is strong support for the existence of a clade including Siettitia, Iberoporus, and Rhithrodytes, what we call the Siettitia group of genera. Our results also clearly demonstrate the parayphyly of Rhithrodytes, and the need to transfer two of the species to maintain its monophyly. The relationships between Rhithrodytes and the other three European stygobiont genera of Siettitiina (Siettitia, Iberoporus, and Etruscodytes), although widely recognised, had not been clearly established. Originally, the genus Rhithrodytes was erected for a group of species of Graptodytes (the group IV of Zimmermann 1919, or the group “crux” of Guignot 1947) with a curved apex of the median lobe of the aedeagus, a lateral stria running the whole length of the pronotum (Bameul 1989) and (as recognised later), a transverse carina in the epipleura (Fery 2013). With the exception of the epipleural carina, the rest of the characters are shared with the subterranean genus Siettitia, which has been for long recognised to be closely related to some of the species included in Rhithrodytes (e.g., R. bimaculatus (Dufour, 1852); Régimbart 1905, Zimmermann 1932) (Table 3).

Subsequent to the description of Rhithrodytes two genera were described each for a single European stygobiont species: Iberoporus and Etruscodytes. Iberoporus cermenius shares the structure of the male genitalia with Rhithrodytes and Siettitia, but it is in particular very similar to that of I. agnus and I. argaensis. These two species (formerly in Rhithrodytes) have a more straight median lobe and a different shape of the apex of the parameres (Bilton and Fery 1996, Fery 2016).

The body shape of I. agnus and I. argaensis has also some similarities to the species of Iberoporus, parallel-sided and elongated (Figs 5b, c; see figs 12–19 in Fery 2016). Iberoporus cermenius shares with Siettitia the structure of the metacoxal processes, something that could be related to the subterranean habitat and a poor swimming ability (Castro and Delgado 2001).

Etruscodytes, described from a male and a female, also shares with Rhithrodytes and Siettitia the general structure of the aedeagus (note that the tip of the aedeagus in the figure of Mazza et al. 2013 seems to be broken) and the long lateral striae of the pronotum (Table 3), but nevertheless was described in a separate genus due to some morphological peculiarities (Mazza et al. 2013). Thus, according to the description by Mazza et al. (2013), the species would have (1) head wide and “subsquare” (regularly rounded in Siettitia and Rhithrodytes; although more similar to that of Iberoporus); (2) presence of short and flattened setae on pronotum and elytra; (3) prosternal process contacting anteromedial process of metaventrite (also in Rhithrodytes, not in Siettitia and Iberoporus, Table 3); (4) anteromedial process of metaventrite rounded (pointed in Siettitia according to Mazza et al. 2013); (5) ventrites II and III not fused (fused in Siettitia and I. cermenius, not in I. pluto sp. n. or Rhithrodytes); (6) elytra not completely fused (fused in Siettitia, not in Iberoporus and Rhithrodytes). Some of these characters seem to be clear autapomorphies related to the subterranean life (fusion of elytra or ventrites, particularly shaped setae, lack of lateral striae on the pronotum, lack of carina on the epipleuron), and others are of uncertain interpretation. Thus, the structure of the prosternal process is sometimes difficult to appreciate, but there do not seem to be fundamental differences between the species (note that in fig. 7 in Mazza et al. 2013 the prosternal process seems to fit below the anteromedial process of the metaventrite, which is likely an artefact), being the differences consequence of the different position of the mesocoxa (contiguous or not) and ultimately the width of the body, which in turn may depend on the habitat and ecology of the species. More data, especially molecular sequences of Etruscodytes and Siettitia, and the likely discovery of other subterranean taxa would contribute to the understanding of the evolution of this western Mediterranean lineage.

We thank all collectors listed in Table 1 for their material, in particular P Richoux (Lyon) for providing specimens of Siettitia avenionensis; A Cardoso and A Villastrigo (IBE) for laboratory work; and H Fery (Berlin) for multiple comments and improvements of the manuscript. Molecular work was partly funded by projects CGL2013-48950-C2-1-P and CGL2016-76705-P (AEI/FEDER, UE) to IR. ASR is supported by a research grant (15471) from VILLUM FONDEN. The holotype was collected under permits of the Instituto de Conservação da Natureza e das Florestas.