Iberobathynella imuniensis Camacho, 1987

Outgroup = Iberobathynella imuniensis Camacho, 1987 ( Parabathynellidae ).

Pleotelson ( Fig. 7I–L View Figure 7 ): Pleotelson seta always dorsal in Bathynellidae , but size variable among species. Very small seta in V. vasconica ( Fig. 7L View Figure 7 ) and much longer in the other Spanish species.

MOLECULAR RESULTS

The concatenated COXI –18S data set is represented by 34 sequences of 1514 bp. Five hundred and eight base pair COXI and 1006 bp 18S sequences were obtained, respectively, from 28 and 31 specimens of Bathynellidae . Best-fit models identified by JModelTest are as follows: COXI TVM + I + G and 18S TIM3ef + I + G according to the AIC and COXI TVM + I + G and 18S TrNef + I + G according to the BIC. The partition scheme suggested by PartitionFinder according to the BIC was to partition both genes and to further partition COX 1 by codon position: COXIPos1 1–508\3, COXIPos2 2–506\3, COXIPos3 3–507\ 3, 18S 509–1514.

The uncorrected sequence divergence estimates for 18S, with maximum and minimum genetic divergences between the subfamilies and with outgroup taxon, are summarized in Table 6. The genetic divergence ranges within the family Bathynellidae are consistent with previous studies ( Camacho et al., 2016). For 18S, sequence divergence between the Bathynellidae subfamilies and the outgroup used in the phylogeny ( Iberobathynella imuniensis of the Parabathynellidae family) ranged between 6.5 and 13.8% ( Table 6), but the sequences analyded have different lengths, and a more complete study could produce different outcomes.

For comparison, genetic divergence for 18S has been examined. Among the genera, 18S divergence ranged from 4.3% (between Gallobathynella and the genus from Texas) to 10.9–14.9% (between the new Australian genus Pilbaranella and the non-Australian genera). Between the two genera of Bathynellinae , genetic distance ranged between 2.3 and 3.5%, the distance between Gallobathynellinae genera was between 6.0 and 9.9% and among the three undetermined Australian genera, genetic distance ranged from 6.2 to 8.7%. The Australian genera were more closely related to the Bathynellinae genera (genetic divergence = 6.2– 8.7%) than to the Gallobathynellinae genera (genetic divergence = 7.2–11.1%). Among genera within the same subfamily, divergence ranges (3.5–6.7%) are consistent with data from previous studies ( Camacho et al., 2013a).

ML (bootstrap support, BS) and BI (posterior probabilities, PP) phylogenetic analyses recovered similar topologies, supporting three monophyletic clades corresponding to Bathynellinae , Gallobathynellinae and presumed Austrobathynellinae ( Fig. 8 View Figure 8 ). The Gallobathynellinae clade includes the genera Vejdovskybathynella , Paradoxiclamousella and Gallobathynella and the undetermined taxa (new genus Camacho et al., in review) from Texas as a sister group (PP = 1). We tentatively consider the Texas taxon as a member of the Gallobathynellinae subfamily. Within Gallobathynellinae , the type genus Gallobathynella (PP = 0.99; BS = 86) was recovered as a strongly supported, monophyletic sister group (PP = 1) with respect to Paradoxiclamousella (PP = 1; BS = 100) and Vejdovskybathynella (PP = 1; BS = 85). All three Gallobathynellinae genera form monophyletic lineages ( Fig. 8 View Figure 8 ), supporting the morphological classification. Within the genus Vejdovskybathynella , there are three well-differentiated groups corresponding to (1) the three well-studied populations (including the type locality) of V. edelweiss in Ojo de Guareña cave and Huesos cave (Burgos); (2) the populations from Redonda Cave (Burgos), Imunia Cave (Burgos) and the Cave of Udias (Cantabria), which are morphologically similar and were initially identified as V. edelweiss but later considered cryptic species according to molecular results ( Camacho et al., 2011); and (3) the Basque populations of V. vasconica and the two populations from Río Chico Cave (Cantabria) and Erizos River in Ojo de Guareña cave (Burgos). The latter two populations were also initially identified as V. edelweiss based on morphology but later identified as a cryptic species through molecular analyses ( COXI ) ( Camacho et al., 2011).

We included four populations of Paradoxiclamousella from the type locality CO69 Cave and CO209 Cave (Asturias), La Nava Cave and Carnero Cave (Cantabria). All specimens were morphologically identified as P. fideli Camacho, Dorda & Rey, 2013 , and the genetic data support most of the identifications except in the case of the population from La Nava Cave, which could represent a cryptic species. More material is needed to confirm the taxonomic status of this lineage.

Within the genus Gallobathynella , the molecular data support the three species identified morphologically ( G. coiffaitti , G. boui Serban, Coineau & Delamare Deboutteville, 1971 and G. tarissei Serban, Coineau & Delamare Deboutteville, 1971 ). It is probable that specimens from the Perles Cave (Cambounes) may belong to a different species, while the population of Lacombe Cave (Cambounes) may belong to G. coiffaitti .

The subfamily Bathynellinae forms a monophyletic clade ( Fig. 8 View Figure 8 ) that appears to be the sister group of Gallobathynellinae (PP = 0.96, BS = 78) although it is not very well supported (BS = 71). Within Bathynellinae the molecular data support the genera identified morphologically and also reflect geographic distribution, with Pacificabathynella from Alaska identified as the sister group to the European genera among which the two species from Slovenia were most closely related.

The third supported clade is composed of the Australian specimens ( Fig. 8 View Figure 8 ). The molecular data seem to reflect geographic distribution in this case too, with three supported lineages from Queensland, Western Australia (Pilbara) and South Australia. These lineages probably represent Austrobathynellinae since the specimen from the Pilbara region exhibits some morphological characters of that subfamily (Perina et al., in press). The lack of molecular data from the three described Austrobathynellinae genera ( Austrobathynella , Transvaalthynella and Transkeithynella ), however, prevents us from confirming the subfamiliar position of the Australian specimens examined in this study.