Racovitzaibathynella dumonti, Camacho & Brancelj & Dorda & Casado & Rey, 2016

Camacho, A. I., Brancelj, A., Dorda, B. A., Casado, A. & Rey, I., 2016, New Parabathynellidae species in Africa: the first bathynellids from Chad and an assay of their phylogenetic position in the order Bathynellacea (Crustacea: Malacostraca) based on 18 S sequences, Journal of Natural History 50, pp. 2691-2726 : 2705-2722

Racovitzaibathynella dumonti sp. nov.

( Figures 4 – 5)

Material examined

Type locality. Dry channel of the river Oued Douar near village Totous, Tibesti area ( Chad) (19°26 ′ 22 ′′ N, 17°31 ′ 47 ′′ E, altitude 585 m; Datum WGS84: 19.43944 N, 17.52972 E, Z 585; 14 March 2014, (16 males, 13 females and eight juveniles); collected by A. Brancelj. All specimens belong to the type series, 25 morphotypes together with DNA extractions from 12 specimens (four females and eight juveniles or subadults) used for molecular analysis, 12 DNA types.

Details of the descriptions are based on adult specimens. The holotype is a male ( MNCN: 20.04/10150), the allotype is a female ( MNCN:20.04/10151) and the morphological type series contains 15 males ( MNCN:20.04/10160 to MNCN:20.04/10174) and eight females ( MNCN:20.04/10152 to MNCN:20.04/10159) . The molecular type series contains five DNA-types ( MNCN:ADN:29981 to MNCN:ADN:29985, MNCN:ADN:29988 and MNCN: ADN:54532 to MNCN:ADN: 54537).

Description

Body. Total length of holotype 1.1 mm and allotype 0.91 mm. Total length of males 0.76 – 1.04 mm, of females 0.72 – 1.03 mm. Body elongated, segments widening towards posterior end; approximately 9 times as long as wide. Head a little longer than wide. Pleotelson with 1 medium-sized plumose ventral seta on each side. All drawings are of the holotype (male) except Th VIII and one figure of labrum and antenna that belong to the allotype (female).

Antennule ( Figure 4a). Six-segmented; length of first 3 segments 1.3 times longer than other 3 segments combined; sixth segment as long as third segment but half as wide; inner flagellum rectangular, large, as large as half the height of the fourth segment; setation as in Figure 4a; segment three with 3 smooth setae; segment five with 2 terminal aesthetascs, similar in size; sixth segment with 3 aesthetascs, each of a different size. A.I longer than antenna.

Antenna ( Figure 4b). Five-segmented; as long as the first 4 segments of A.I; first 2 segments shortest; third and fifth segments similar in size and slightly longer than the fourth segment; last segment with 3 setae: 1 is smooth, 1 plumose and the outer seta transformed into a long strong fang curved inwards (sexual dimorphism); setal formula: 0/0/1+0/1+0/3(1).

Antenna of the allotype ( Figure 4c). Similar to that of holotype but the outer seta of last segment smooth and not transformed; setal formula as in holotype.

Labrum ( Figure 4d). Distal edge almost flat, with 10 teeth, 6 central conical and 2 lateral teeth at each side, sub-triangular, slightly smaller than the central teeth.

Labrum of the allotype ( Figure 4e). Similar to the male but with fewer teeth, only 8 in total: 6 central teeth and o1more lateral tooth at each side.

Mandible ( Figure 2e ). Pars incisiva with 4 teeth on distal part, stout and strong tooth on the ventral edge; pars molaris with 4 claws, 2 joined proximal claws with a large number of fine hairs; mandibular palp 1-segmented, with a distal seta as long as the pars incisiva.

Maxillule ( Figure 4g). Proximal endite with 2 long claws with long setules at base and short setules on the rest and 2 more small claws also with setules; distal endite with 5 smooth claws, 3 grouped in distal part of endite, and with 3 subterminal smooth setae on outer distal margin, unequal in length.

Maxilla ( Figure 1g ). Three-segmented; basal segment almost 2 times shorter than the second segment, armed with 2 smooth setae; second segment twice as long as wide, with 10 setae (3+1 on medial region) and very small third segment with 1 terminal strong claw and 4 setae. Setal formula: 2/10/5 (1).

Thoracopods I to VII ( Figure 5a–g ). Well developed, length gradually increasing from I to VI, Th VI to VII similar in size; epipod absent on Th I and II, present on Th III to VII, almost half the length of basipod; basipod with 1 smooth seta at distal inner corner in Th I to V, absent on Th VI and VII. Exopod 2-segmented, a little shorter than the first 3 segments of endopod on all Ths; first segment long, similar to the 2 first segments of endopod with 2 setae, 1 barbed and 1 plumose on all Ths; segment two about 1.5 times longer than segment one, with 2 terminal setae, 1 barbed and 1 plumose, similar in size to Th I and of different size on the rest of exopod of Ths; all Ths with group of ctenidia at the base of setae. Endopod 4-segmented; the first segment is similar in all Ths, about 2/3 as long as segments two and three in Th I and with a smooth seta ( Figure 5a ), about 1/2 as long as following segments two and three in Th II ( Figure 5b ), less than 1/2 as long as segments two and three in Th III ( Figure 5c ), almost 2/5 as long as segments two and three in Th IV to V and 2/5 as long as segments two and three in Th VI and VII; segments two and three are similar in length in all Ths; the fourth segment is small, with 2 similar claws and 1 smooth seta on Th I and 1 barbed claw and 1 seta on the rest of Ths; Th I to VII with a plumose seta on segment two and 1 small seta on segment three, both on the distal outer corner (see figures); second and third segment with groups of ctenidia which extend almost the entire length of segment Th II to VII.

Setal formulae of endopod: Th I, 1+0/0+1/0+1/3(1); Th II – VII, 0+0/0+1/0+1/2(1).

Male thoracopod VIII ( Figure 4i–l). Rectangular; with penial lobes and outer lobe located on distal quarter of thoracopod; dentate lobe (D. lb) with big teeth; outer lobe (O. lb) fused with basipod (Bsp), distal end almost square, not covering posterior part of D. lb, but covering a big part of exopod (Exp) and distal end of basipod; posterior lobe (P. lb) covers one part of D. lb on caudal face; exopod well developed, individualised, with main axis (long branch) almost parallel to distal edge of basipod and with pointed distal end; the basipod is well developed on the latero-external face, without seta, with distal end bilobed; reduced endopod (Endp), only present as 2 unequal smooth setae located at a shallower level than the posterior region of exopod on the external half of the distal edge of basipod.

Thoracopod VIII of the allotype ( Figure 4m, n). Very reduced, twice as long as wide, trapezoidal, with dilated basis and rounded distal part.

Pleopods. Absent.

Uropods ( Figure 5h ). Sympod 3.7 times as long as wide, 1.5 times as long as endopod and 1.9 times as long as exopod; 7 barbed spines of similar size, located at the distal half of sympod, smaller than the apical spine of the endopod; endopod 1.3 times longer than exopod and twice as wide, with a long apical spine with a membrane formation in distal half with setules and 4 setae, 2 terminal barbed and 2 latero-external plumose setae of similar length, with 4 groups of strong ctenidiae on dorsal face; exopod has 2 terminal barbed setae, of very different lengths, 1 very small.

Uropod of allotype. Similar to holotype, except for the number of spines on sympod, which had 5.

Pleotelson ( Figure 5i ). With 1 small, plumose ventral seta on either side near the base of the furca. Anal operculum not protruded.

Furcal rami ( Figure 5i ). Rectangular, 1.7 times as wide as long, with 3 strong barbed spines, the distal 1 slightly longer than the others, similar in length; at the base of the spines a membrane with setules in the distal edge; on the dorsal side of furcal rami are the setae characteristic for members of the family Parabathynellidae , plumose and different in length.

Variability

The observed variability includes only the number of spines on sympod of the uropod, varying between five and eight.

Etymology

The species ‘ dumonti ’, is named as a tribute to the professor Henry Dumont (University of Ghent, Belgium), organiser of the Chad 2014 expedition and well-known expert in limnology of Sahara.

Remarks

According to observed characters, the new species belongs to the genus Racovitzaibathynella and not to the genus Cteniobathynella , although both genera are closely related. The main differences between this genus and Cteniobathynella , and all other African genera, were pointed out by Serban and Coineau in 1994, when described Racovitzaibathynella . Regarding the secondary traits, all African genera are similar. However, there are clear strong differences in the male Th VIII, as well as in the sexual dimorphism present in the antenna ’ s chaetotaxy, which only occurs in Racovitzaibathynella . This third species of the genus shares the presence of teeth (absent in Cteniobathynella ) in the penial region of the male Th VIII, the specific position of the setae in the endopod, where this seta is all that remains of the endopod (there is a small endopod merged to the basipod in Cteniobathynella ), and in the presence of a posterior lobe. All of these traits locate this species within Racovitzaibathynella . According to Serban, the sexual dimorphism expressed in the chaetotaxy of the antenna is also a defining trait of the genus.

The male thoracopod VIII is not well known in most species of Cteniobathynella , but the opposite is true for the two species of Racovitzaibathynella , which allows us to make a good comparison between them and our specimens.

The new species is of a similar size to the other two known species of the genus, and differs from them in the traits detailed in Table 1, some of which are discussed next. R. dumonti sp. nov. has fewer setae in the first three segments of A.I than R. emilei does; in the new species there are no setae in the second segment of A.II as in the case of R. emilei and R. transvaalensis ; the labrum in the new species has fewer teeth and is not as flat as in the other species. The rest of the mouth parts show minimal differences. The new species lacks the epipod in the thoracopods I and II, while in the other species the Th II does have an epipod and lacks setae in the basipods of the thoracopods VI and VII, which is present in the rest of its congeners; the endopod of all the thoracopods in the new species has a higher number of ctenidia than that of the rest of the species of the genus; the segment size proportion of the exopods and endopods in all thoracopods is similar in all three species. The sympod of the uropod in the new species shows spines of the same size, while in the other species the distal spine is larger than the rest; the furcal ramus of the new species is rectangular like in the other species, but it is thicker and less elongated than in its congeners. The female thoracopod VIII of the new species is not as oval as in the other species, being more trapezoidal and with a conical tip. In regards to the male thoracopod VIII, the general aspect and position of the lobe is similar, in agreement with the generic description, although there are some differences in regards to the other two species. The outer lobe is almost square, instead of triangular, and does not cover the posterior part of the dentate lobe; the dentate lobe has fewer teeth; the posterior lobe is longer; the basipod has two lobes in the distal region and the two setae of the endopod are different sizes, while in the other two species they are longer.

Molecular results

We have obtained nuclear DNA sequences of 12 specimens of Racovitzaibathynella dumonti sp. nov. and two specimens of Haplophallonella irenae sp. nov. (see Table 2 for GenBank accession numbers). They were compared with representatives of other taxa from the families Bathynellidae (four genera and 12 species) and Parabathynellidae (five genera and 17 species) from USA, Spain and France. With these data and the sequences obtained from GenBank for four Australian genera (11 species), we made a preliminary phylogenetic analysis to assess the position of the two new species from Chad in regards to the genera of the world for which nuclear DNA sequences are published – in total 13 – at the time of this analysis.

Nucleotide analysis

The alignment of all Bathynellacean 18S rRNA gene sequences, 45 specimens in the case where Australian and US species are included (709 bp), comprised 38% variable sites and 32 specimens (1333 bp); without Australian species, it comprised 54% variable sites; the base frequencies were similar in the two cases, between 22 and 28% (24.6 – 25% A, 25 – 23% C, 28 – 27% G and 22 – 24% T) .

Genetic divergences

The uncorrected sequence divergence estimates of 18S rRNA between all the genera studied including Australian and USA genera are summarised in Table 3, and in Table 4 excluding these genera. The values are generally higher when we include the Australian species in the analysis, as a smaller number of base pairs are used. The values for the comparisons are more appropriate with more base pairs analysed, providing a more realistic picture of the similarities and differences between the genera. All Australian genera belong to the Parabathynellidae family and the maximum genetic divergence with the outgroup Anaspides tasmaniae is shown by Hexabathynella (12.6%), while the minimum is shown by the genus Atopobathynella (10%). The Australian genera show a higher genetical divergence with Racovitzaibathynella and Haplophallonella than with the Spanish genera, and a higher divergence in particular with Haplophallonella (10.5 – 12.7%) than with Racovitzaibathynella (8.7 – 11.3%). The Australian Hexabathynella shows a very high divergence of 8.3% with the Spanish Hexabathynella , even higher than it shows in regards to other Spanish genera like Iberobathynella or Paraiberobathynella , 6.6% and 6.7%, respectively. In regards to other Spanish genera, the smallest divergence is shown with a species of Atopobathynella (3.7%) and the largest with Octobathynella (7.9%); the species of Brevisomabathynella and Billibathynella present values between 5.5% and 7.2% with the species of the Spanish genera. The only specimen of US Bathynellidae studied presents a divergence of 10.4% with the outgroup, and the divergence is also high (9.6%) in regards to the Spanish genus in the family Paradoxiclamousella , while it is relatively low in regards to Vejdovskybathynella and Gallobathynella , ranging between 4.6 and 5.4%. When the Australian and US species are removed, the genetic divergence between the outgroup and the genera of both families of Bathynellacea ranges from 9.3 to 13.1%. Anaspides shows greater genetic phylogenetic analysis.

(Continued) divergence with Bathynellidae genera than with the Parabathynellidae genera studied: 9.4 – 13.1% with three Bathynellidae genera and 9.3 – 12% with five Parabathynellidae genera. The highest genetic divergence is shown with the genus Haplophallonella (13.1%), while the value ranges between 11.9 and 12% with the four specimens studied of Racovitzaibathynella dumonti sp. nov. The smallest divergence is shown with species from the genus Iberobathynella (9.3%) and the genus Gallobathynella (9.4%), each from a different family.

Within the Bathynellidae we have studied three genera, within which the only Paradoxiclamousella species studied (from Spain) shows the same genetic divergence ranging from 4.7 to 5.3% with the five species of each of the other two genera, Vejdovskybathynella ( Spain) and Gallobathynella ( France) . Gallobathynella and Vejdovskybathynella show much less genetic divergence between them (1.2 – 2.3%).

Within the Parabathynellidae , in addition to the two new African species, we included 15 species belonging to three genera (11 species of Iberobathynella , three of Paraiberobathynella and one of Hexabathynella ) (see Table 2), all collected in Spain, with the exception of the African Paraiberobathynella cf. maghrebensis . Racovitzaibathynella dumonti sp. nov. shows the highest genetic divergence with Hexabathynella , and the lowest with the other new African species Haplophallonella irenae sp. nov. (7.1 – 7.2%). The latter also shows the highest genetic divergence with Hexabathynella (12.3%). In regards to the other two Spanish genera, the two new species show similar divergence values, athough these are somewhat lower in the case of Racovitzaibathynella dumonti sp. nov. (9.0 – 10.9%) than in Haplophallonella irenae sp. nov. (10.5 – 11.7%). Iberobathynella and Paraiberobathynella have a relatively high genetical divergence with Hexabathynella , ranging between 6.3 and 7.6%, but this is still lower than what the new species show. Within the genus Iberobathynella , the divergence between species ranges from 0.1% for I. sp. 2 and I. sp. 4, to 3.1% for I. celiana Camacho, 2003 and I. burgalensis Camacho, 2005 . Iberobathynella shows less divergence from some species of the genus Paraiberobathynella (0.3 – 2.6%) than the values between some of the species within the genus (this case requires a thorough study). The maximum divergence of the three Paraiberobathynella species can be found between the Spanish species and the Moroccan species (0.2%).

Phylogenetic analyses

The results of the phylogenetic analyses (ML, Bayesian) are summarised in Figures 6 (all genera) and 7 (without Australian and US genera).

The 18S rRNA sequence data analysis, in both cases, produced trees ( Figures 6 and 7 ) in which there are two groups supported in ML and Bayesian analyses (> 100% Bayesian posterior probability, BPP, and 99% and 100% bootstrap values). All samples of Bathynellidae are clearly separated from the Parabathynellidae .

The phylogenetic reconstruction of Bathynellidae when using all of the genera ( Figure 6 ) shows two well-supported groups (99% bootstrap and maximum BPP): one with the genus from the USA and another with the European genera; within the latter Paradoxiclamousella is clearly separated from the other two genera, which remain scattered in a group composed on one side by the species of Vejdovskybathynella and an undetermined French species of Gallobathynella , but lacking strong support (0.56 BPP and 86% bootstrap), and on the other hand a group composed by the other four species of Gallobathynella , but which lacks structure. The phylogenetic reconstruction of this family has a better structure, with groups better supported, when the analysis is done only with the species for which there are longer sequences, excluding the genus from the USA. In this case ( Figure 7 ) there are three groups (100% bootstrap values): one composed of Paradoxiclamousella and four species of Vejdovskybathynella ; another composed of the fifth Spanish Vejdovskybathynella species and an undetermined French species initially assigned to Gallobathynella ; and a third group which contains the other four French Gallobathynella species.

When analysing the phylogenetic reconstruction of the Parabathynellidae including the Australian species ( Figure 6 ), we see on one side how the new African species remain clearly separated and in two different groups (two genera), while on the other side the two Hexabathynella species (one Spanish, one Australian) are separated from the rest, and each of them is separated with maximum bootstrap values and BPP. Atopobathynella could be considered the sister genus of the rest of the Australian genera ( Brevisomabathynella species and the genera Billibathynella and Octobathynella ), excluding Hexabathynella (although this is poorly supported). The other group is composed of two subgroups, one with the Iberobathynella species from Andalusia ( I.celiana and I. andalusica Camacho ), and another with the rest of the Iberobathynella species plus three Paraiberobathynella . In summary, we can say that they follow a genus structure, each of them grouping the different species studied and assigned to these genera based on the morphological traits.

When analysing the phylogenetic reconstruction of the Parabathynellidae without the Australian species ( Figure 7 ), the group structure appears slightly modified: in one of the groups we find the two African genera containing our newly described species, with a 100% bootstrap value and maximum BPP value, and in another we find Hexabathynella on one side and all the species of Iberobathynella and Paraiberobathynella on the other (66% bootstrap). The three species of Paraiberobathynella compose a subgroup (61%) within a group that included I. cantabriensis as well as other undetermined Iberobathynella species , although these are not too well supported (61% bootstrap).

General discussion and conclusion

In this study we have complemented a morphological analysis with a molecular analysis. We present all of the morphological characters needed to establish a new taxon, as required by classical taxonomy, but we also have added new characters, 18S rRNA sequences, obtained with molecular techniques, as this gene has proved useful in examining higher level crustacean relationships ( Giribet and Ribera 2000).

Partial sequences of 18S rRNA (1333 bp) have been obtained in this study for 32 specimens of 26 species of eight genera from two families. This is the first time this has been done in French Bathynellacea (genus Gallobathynella ) and Parabathynellidae from Africa (three genera). Abrams et al. (2012) obtained partial sequences (about 700 bp) of several species belonging to three Australian genera of Parabathynellidae , and one more sequence of one unassigned specimen of Australian Bathynellidae has also recently appeared in the GenBank sequence of Octobathynella peelensis Camacho and Hancock, 2010 from Australia ( Abrams et al. 2012). They found on average 18S rRNA sequence divergences among genera of between 3.1 and 8.8%, and an average divergence among all Australian parabathynellid species of 4%. In our study, the genetic divergences observed in partial 18S rRNA sequences between the new species and other Parabathynellidae ranged from 8.7 to 12.7% (see Table 3), and among them the divergence is 8.8%. Nevertheless, excluding these two African genera to which the new species described here belong, the divergence we observe between the Iberian genera, the Australian genera and the cosmopolitan Hexabathynella is around 3.7% ( Paraiberobathynella – Atopobathynella ) and 10% ( Brevisomabathynella – Hexabathynella from Spain). Within the Spanish genera, Iberobathynella and Paraiberobathynella show 8.7 – 9.2% divergence with Hexabathynella , and a maximum of 2.1% between them. The Australian genera among them show a divergence between 3.1% ( Brevisomabathynella – Billibathynella ) and 8.7% ( Octobathynella – Hexabathynella ). Among the five species of Australian Brevisomabathynella , the divergence found ranges between 0.1 – 1.6%, and between the two Atopobathynella we find a divergence of up to 3.3%. Within the Iberobathynella genus, the 11 species studied show divergences between 0.3 and 2.4% using short squences, and between 0.1 and 3.1% when using long sequences. In any case, the number of species and genera of Bathynellacea that have been analysed at a molecular level around the world still remains low, and the same is true for the number of genes studied (18S, 16S, cytochrome oxidase 1); thus, it is still early to try to establish the threshold of genetic divergence between and within genera.

Fortunately, in this work the taxonomic position of the two new species seems clear based on our detailed morphological analysis. The molecular data obtained from both genera, for which there was no previous molecular data until now, are an added value that will be especially useful in the future when comparing new discoveries of these or other African genera, as well as from all around the world.

The application of traditional taxonomic tools together with molecular techniques already allow us to have enough elements for comparison to help us make decisions of a taxonomic nature, especially now that more and more cryptic species are being found within this group ( Camacho et al. 2011, 2012, 2013a; Cook et al. 2012) where morphology-only studies lead to underestimating the global diversity of Bathynellacea .

Distribution of Bathynellacea from Africa and Madagascar

Thirty-four species, currently known for the African continent and Madagascar, including the two new ones described here, are listed below. The list includes the type locality for each of them, the date of collection and the name of the collector(s).

Family Parabathynellidae Noodt, 1965 .

(1) Paraiberobathynella maghrebensis ( Boutin and Coineau, 1987) . Well P4, Haouz plain, Marrakesh, Morocco (lying at 3.5 km north of the well P2 which is situated on the terrain of the Science Faculty of the University of Marrakesh; information from Schminke 2011). Leg.: C. Boutin and Boulanouar (1984). Other locality: well, route Nador to Berkane, Maghreb, Morocco (Leg.: D. Jaume, 6 June 2012).

(2) Hexabathynella pauliani ( Delamare Deboutteville, 1953) . Maroantsetra city, three localities: east coast of lagoon; wells of Dr. Prochazka, west of the lagoon and wells of the sawmill Galois, groundwater below littoral border), Madagascar. Leg.: R. Paulian (February 1952).

(3) H. africana Cho and Schminke, 2006 . Olifants River , 400 m above the bridge Citrusdal, Kaapprovinsie, South Africa . Leg .: H.K. Schminke (25 July 1973) .

(4) H. monoaesthetasca Cho and Schminke, 2006 . Olifants River, 400 m above the bridge Citrusdal and on street between Citrusdal and Clanwilliams and 1.25 km above the bridge Citrusdal (26 September 1973), Kaapprovinsie, South Africa. Leg.: H.K. Schminke (25 July 1973).

(5) Notobathynella lemurum Drewes and Schminke, 2007 . Manakaralahy River, 150 km above the ford of road N 10, Madagascar. Leg.: H.K. Schminke (15 September 1974).

(6) Cteniobathynella essameuri Dumont, 1981 . Spring Ain Essameur, on the flanks of Djebel Idjerane, is part of the Adrar Ahnet, some 150 km south-east of In Salah, Central Algeria and Central Sahara, Algeria. Leg.: H.J. Dumont? (biological expedition 13 September 1979).

(7) C. bakeri ( Green, 1964) . Kaiso Spit, Lake Albert, Uganda. Leg.: J. Green (October 1962).

(8) C. caparti ( Fryer, 1957) . Sandy beach at Samfya, near the south-west corner of Lake Bangweulu, Zambia; several rivers (Shewanoe River, streams of Matopo Hills, Bembezi River, Nuanetzi River) found by Wells, 1964, Zimbabwe. Leg.: G. Fryer (March 1956, September 1956).

(9) C. leleupi Delamare Deboutteville and Chappuis, 1955 . Uvira, north-west corner of Tanganyika Lake, Democratic Republic of Kongo. Leg.: N. Leleup (7 July 1954).

(10) C. teocchii ( Coineau et Knoepffler, 1971) . Vivid stream at the exit of a cave in the savanna Bébé about 3 km from Boukoko, near M ’ Baiki in the Lobaye valley, Central African Republic. Leg.: L.-Ph. Knoepffler (23 September 1969).

(11) C. calmani ( Por, 1968) . En Nur, Spring 6 in the Tabgha group of springs, Heptapegon, En Nur, near the northern shore of Lake Tiberias, Lower Galilee, Israel. Leg.: M. Tsurnamal (1967).

(12) Haplophallonella heterodonta Serban and Coineau. Bandama River near Lamto, Ivory Coast. Leg.: L.-Ph. Knoepffler (27 July 1966).

(13) Haplophallonella irenae sp. nov. Ouet Douar River , near Totous village, Tibesti Mountains, Chad . Leg .: A. Brancelj (14 March 2014) .

(14) Racovitzaibathynella emilei Serban and Coineau, 1994 . Mutlumuvi River, tributary of the Sabie River, Kruger National Park (16 miles from Skukuza), Transvaal, South Africa. Leg.: Y. Coineau and L.-Ph. Knoepffler (3 February 1972).

(15) Racovitzaibathynella transvaalensis Serban and Coineau, 1994 . Mutlumuvi River, tributary of the Sabie River, Kruger National Park (16 miles from Skukuza), Transvaal, South Africa. Leg.: Y. Coineau and L.-Ph. Knoepffler (3 February 1972).

(16) Racovitzaibathynella dumonti sp. nov. Ouet Douar River , near Totous village, Tibesti Mountains, Chad . Leg .: A. Brancelj (14 March 2014) .

(17) Ctenophallonella mutlumuviensis Coineau and Serban, 1978 . Mutlumuvi River, tributary of the Sabie River, Kruger National Park (16 miles from Skukuza), Transvaal, South Africa. Leg.: Y. Coineau and L.-Ph. Knoepffler (3 February 1972).

(18) Habrobathynella jeanneli ( Delamare Deboutteville and Paulian, 1954) . Lanirano lagoon near Tolagnaro ( Fort Dauphin ), Madagascar . Leg.: R. Paulian (April 1953) .

(19) Habrobathynella milloti ( Delamare Deboutteville and Paulian, 1954) . Maroantsetra city, east coast of lagoon and wells of the sawmill Galois, groundwater below littoral border), Madagascar. Leg.: R. Paulian (February 1952) .

(20) Heterodontobathynella ninianae ( Green, 1964) . Kaiso Spit, Lake Albert, Uganda. Leg.: J. Green (November 1962).

(21) Thermobathynella adami Capart, 1951 . Hot spring of Kaziba on the left bank of the River Senze, tributary of River Lufira (Upemba National Park), Katanga, Democratic Republic of Congo. Leg.: W Adam (15 April 1949).

(22) Nilobathynella predinastica Dumont, 1984 . Station near south end of Lake Nubia, close to the village of Abri, Nubia, Sudan. Leg.: H.J. Dumont (March 1982).

(23) Afrobathynella trimera Schminke, 1976 . Koega River , not far from Smitskraal, along the road Patensie-Willowmore, Eastern Cape, South Africa . Leg .: H.K. Schminke , (8 August 1974) .

(24) Nunubathynella dimera Schminke, 1976 . Koega River , not far from Smitskraal, along the road Patensie-Willowmore, Eastern Cape, South Africa . Leg .: H.K. Schminke , (8 August 1974) .

Family Leptobathynellidae Noodt, 1965

(25) Leptobathynella gigantea Schminke 2011 . Olifant River, near Balule, Transvaal, Kruger National Park, South Africa. Leg.: H.K. Schminke (27 July 1973).

(26) Parvulobathynella duodecima Cho and Schminke, 2001 . Olifant River , 100 m above the bridge of the road between Clanwilliams and Klawer, Kaapprovinsie, South Africa . Leg.: H.K. Schminke (26 June 1973).

(27) P. octacantha Cho and Schminke, 2001 . Sandspruit, about 12 km from Utrecht, on the junction of R 27 to Dundee , Natal, South Africa . Leg.: H.K. Schminke (5 August 1973).

(28) Lamtobathynella pentodonta Serban and Coineau 1982 . Nizi River between Ndouci and Lamto, Ivory Coast. Leg.: L.-Ph. Knoepffler (6 September 1969).

(29) Acantobathynella knoepffleri Coineau, 1967. Bandama River at Lamto, Ivory Coast. Leg.: L.-Ph. Knoepffler (27 July 1966).

Family Bathynellidae Grobben, 1905

(30) Nannobathynella eburnea Schminke, 1979 . Sassandra, route of Worofla to Touba, Ivory Coast. Leg.: B. Statzner (22 April 1977).

(31) Nannobathynella africana Schminke and Wells, 1974 . Nuanetzi River, Zimbabwe. Leg.: Wells, 1964? Other localities: Tsende River, Kruger National Park, South Africa.

(32) Agnatobathynella ecclesi Schminke, 1980 . Mwanza River , route Blantyre-Chiromo, Malawi . Leg .: D.H. Eccles (17 October 1973) .

(33) Transvaalthynella coineaui Serban and Coineau. Mutlumuvi River , tributary of the Sabie River, Kruger National Park (16 miles from Skukuza), Transvaal, South Africa. Leg.: Y. Coineau and L.-Ph. Knoepffler (3 February 1972).

(34) Transkeithynella paradoxa Serban and Coineau. Umzibuvu River, Transkei , South Africa. Leg.: Y. Coineau and L.-Ph. Knoepffler (6 February 1972).

In a continent as large as Africa, only 29 species are known, as well as four described from Madagascar ( Schminke 2011) and one from Israel, and all of them come from just a few samples from very small areas. The sampling effort has been very limited and only opportunistic, in less than a fourth part of the 54 countries that compose the continent: Morocco, Algeria, South Africa, Uganda, Ivory Coast, Zimbabwe, Malawi, Zambia, Sudan, Democratic Republic of Congo, Central African Republic and Chad. It is remarkable that in a single sample up to four different species have been found (e.g., in Mutlumuvi River Transvaalthynella coineaui , Ctenophallonella mutlumuviensis , Racovitzaibathynella emilei and R. transvaalensis were found; and in Olifant River Leptobathynella gigantea , Parvulobathynella duodecima , Hexabathynella africana and H. monoaesthetasca ) and in most of the samples where abundant material appears at least two species have been found (e.g., in Koega River Afrobathynella trimera and Nunubathynella dimera or Haplophallonella irenae sp. nov. and Racovitzaibathynella dumonti sp. nov. in Ouet Douar River). This leads us to think that the number of species left to discover and describe is very high due to the extent of the territory. As a matter of fact, we are aware that many species are already being studied, although they may have not been formally published, because the doctoral thesis of Cho (1995) mentions at least nine Leptobathynella species new to science, eight from South Africa and one from Malawi, and all of them found in samples collected in the past, some of which have also produced the abovementioned species.

Figures

Figure 1. Racovitzaibathynella dumonti sp. nov. Male habitus; 10× microscope photography (MNCN).

Figure 2. Haplophallonella irenae sp. nov. (a–l). Male holotype. (a) Antennule (dorsal view); (b) antenna (dorsal view); (c) labrum; (d) allotype female labrum; (e) mandible; (f) maxillule; (g) maxilla (dorsal view); (h) thoracopod VIII (latero-external view); (i) thoracopod VIII (latero-external view); (j) thoracopod VIII (latero-internal view); (k) thoracopod VIII (latero-internal view); (l) uropod (lateroexternal view). Abbreviations: D. Lb, dentate lobe: I. Lb, inner lobe; O. Lb, outer lobe; Bsp, basipod; Exp, exopod; B.reg, basal region; P.reg, penial region. Scale bars in mm.

Figure 5. Racovitzaibathynella dumonti sp. nov. (a–i) Male holotype. (a) Thoracopod I; (b) thoracopod II; (c) thoracopod III; (d) thoracopod IV; (e) thoracopod V; (f) thoracopod VI; (g) thoracopod VII; (h) uropod (latero-external view); (i) furcal rami (dorsal view). Scale bars in mm.

Figure 6. Phylogenetic reconstruction of the 18S rRNA data of Bathynellacea of the world. Posterior probability Bayesian consensus tree with re-estimated branch lengths using the GTR + I + G model of substitution. Bayesian posterior probabilities and credibility values are shown above on corresponding nodes, and bootstrap values (ML) below.

Figure 7. Phylogenetic reconstruction of the 18S rRNA data of European and African Bathynellacea. Posterior probability Bayesian consensus tree with re-estimated branch lengths using the GTR + I + G model of substitution. Bayesian posterior probabilities and credibility values are shown above on corresponding nodes, and bootstrap values (ML) below.