Between 2019 and 2021, 5 sampling campaigns (3 during the rainy season and 2 during the dry season) were carried out in 41 wells in the city of Douala. The wells were located in alluvial sediments near the Atlantic coast (maximum distance from the ocean 16 km) with an elevation lower than 65 m a.s.l., and a maximum depth of 9 m. The city of Douala has warm and humid climate conditions, with an average annual temperature of 27.0 °C and an average humidity level of 83%, with around 4,000 mm of precipitation per year (Olivry 1986). The dry season extends from December to February and a long rainy season extends from March to November.

Faunal samples were collected from the bottom of the wells using a modified phreatic net sampler (30 cm diameter aperture and 64 μm mesh size: Cvetkov 1968).

Water samples were collected in each well at each sampling date. Temperature, pH, electrical conductivity, and dissolved oxygen were measured directly in the field with a mercury thermometer, a portable pH-meter (CG 818, Schott instruments), and a portable conductivity meter and optical dissolved oxygen meter (HQ30d, Hach Lange), respectively. . The water was transported directly to the laboratory using polyethylene bottles under cool storage for additional analyses: alkalinity, dissolved carbon dioxide, calcium, magnesium, chemical (CDO) and biological (BDO5) oxygen demand, turbidity, and nutrient contents. The differences in physico-chemical parameters of water in wells with or without the new species were assessed using non-parametric Mann-Whitney U Tests using the STATISTICA v12 (StatSoft) software.

The specimens were dissected and mounted on microscopic slides in Faure’s mounting medium, after maceration +in lactic acid and staining with pink lignin. Body parts were digitally drawn using a Wacom tablet and the Adobe Illustrator software package (Adobe).

Total genomic DNA was extracted from a part of an animal using NucleoSpin Tissue Kits (Machery-Nagel) following the manufacturer’s instructions (Düren, Germany). A fragment of the mitochondrial COI gene was amplified using LCO1490/HCO2198 (Folmer et al. 1994) and UCOIR/UCOIF (Costa et al. 2009) primers. Touchdown Polymerase chain reactions (TD PCRs) (Korbie and Mattick 2008) were performed in a final volume of 27 μl containing 12 μl of water, 10.5 μl of Type-it PCR Master Mix (Qiagen, Germany), and 1.8 μl of each primer (5 μM). A denaturation step at 95 °C for 5 minutes was followed by 35 cycles (30 seconds at 95 °C, 90 seconds at each temperature and 30 seconds at 72 °C), with a final extension step of 30 minutes at 70 °C. TD-PCRs are characterized by an initial annealing temperature (55 °C in our study) above the projected melting temperature (Tm) of the primers, and then progressive transitions to a lower, more permissive annealing temperature over the course of the successive cycles (-0.5 °C per cycle during the first 8 cycles). The next 27 cycles were performed at 51 °C.

The PCR results were checked by gel electrophoresis, then the PCR products were purified and sequenced in both directions by the Eurofins sequencing facility or with an Applied Biosystem 3130 XL sequencer in the DNA sequencing facility of the Institute of Genetics and Development of the University of Rennes (https://igdr.univ-rennes1.fr/en). All COI sequences obtained in four wells are deposited on Genbank (assession numbers OL514108; OL514109; OL514110; OL51411 and OL51412) and on the Barcode of Life Data systems (BOLD) (BOLD process id: METAF001-21, METAF002-21, METAF003-21, METAF004-21 and METAF005-21).

The new COI sequences were supplemented by COI sequences downloaded from Genbank: two sequences of Metastenasellus species from Benin (accession numbers KY623773.1 and KY623774.1); four from M. camerounensis from Cameroon (accession numbers KY623769.1; KY623770.1; KY623771.1; KY623772.1), and two from an unknown Metastenasellus species from Cameroon (accession numbers KY623775.1; KY623776.1). All sequences were aligned with the MUSCLE algorithm (Edgar 2004) implemented in SEAVIEW ver. 5 (Gouy et al. 2021)to explore the Metastenasellus species diversity using different species delimitation methods implemented in iTAXOTOOLS 0.1 (Vences et al. 2021). Firstly, according to distance-based methodologies: the Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al. 2021) and the distance-based Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012). Secondly, the results of the genetic distance-based species delimitation were cross-validated with the phylogenetic tree-based delimitation methods, namely: Generalized Mixed Yule Coalescent (GMYC) (Pons et al. 2006) and multi-rate Poisson tree processes (mPTP) (Kapli et al. 2017). For tree-based species delimitation methods, we built a phylogenetic tree under a maximum likelihood framework (ML) using PhyML v3.1 algorithm (Guindon et al. 2010) implemented in SEAVIEW ver. 5 (Gouy et al. 2021). For the tree, we used HKY85+I model, selected as the best-fit model of evolution with jModelTest 2 (Darriba et al. 2012).

For final visualization with already sequenced species of Metastenasellus, the neighbor-joining tree of all COI sequences, using the Kimura two-parameter (K2P) model of evolution (Kimura 1980) with 1000 bootstrap replicates, was created in SEAVIEW ver. 5 (Gouy et al. 2021).

An exhaustive survey of the literature on Stenasellidae (species descriptions, identification keys, monography, PhD theses, books, papers) was carried out for all the species known in continental Africa. All maps were drawn with Arcgis Desktop 10.4 software (Esri) and using GIS data available on DIVA website (http://www.diva-gis.org) in WGS 84 datum.

The new species differs from most of species of Metastenasellus by the presence of an external lobe on the protopodite of pleopod 2 which is known only in Metastenasellus leysi Magniez, 1985. However, M. boutini differs from M. leysi by many other characteristics such as the total length (< 3.5 mm for M. leysi); the number of articles on flagellum of antenna 1 and 2 (2 and 13, respectively) much reduced for M. leysi in comparison with M. boutini (15 and 48, respectively); pleonites 1 and 2 as long as pereonite 7 for M. leysi (as long as 0.5 fold pereonite 7 for M. boutini). The shape of the endopodite of pleopod 2 of M. boutini is also characterized by a distal part of spermatic duct slightly protruding out the second article. By these characteristics, the new species strongly differs from M. camerounensis, M. leleupi (Chappuis 1951), M. dartivellei (Chappuis 1952), M. congolensis (Chappuis 1951), and M. powelli Magniez 1979. By its spermatic duct, M. boutini resembles to M. wikkiensis Lincoln 1972 and M. tarrissei Magniez 1979. It differs from M. wikkiensis by a shorter size of the uropod, as long as 50% the pleotelson for M. boutini (as long as 2 fold the length of the pleotelson in longest specimens of M. wikkiensis) and by the absence of the terminal setae on the second article of the exopodite of pleopod 2 (terminal setae present in M. wikkiensis). M. boutini differs from M. tarrissei by the total length and the number of article on flagellum of antenna 1 (8 for M. tarrissei and 15 M. boutini); pleonites 1 and 2 shorter than pereonite 7 for M. boutini (as long as the length of pereonite 7 for M. tarrisei).

We sequenced and analyzed DNA from five individuals from four wells, including the type locality at Douala. Based on the Folmer’s fragment of COI marker, the new species is clearly distinct from the other species sequenced in Cameroon and in Benin. The pairwise genetic distances between M. boutini and all other species varied between 22.4 and 27.8% for Cameroonian species and even 28.2% for the species in Benin (Fig. 5). In addition to genetic distance and morphological distinctness, all delimitation methods clearly highlighted the existence of at least five distinct lineages of Metastenasellus in the material available for Africa (Fig. 5). All delimitation methods confirmed that individuals from wells at PK21 belong to the same lineage. Consequently and in addition to morphological distinctness, molecular data support the hypothesis that M. boutini can be considered as a new species, which strongly differs from M. camerounensis and the two other species already sequenced in Cameroon indicated as Metastenasellus sp1 and sp2.

Figure 5. 

Neighbor-joining tree of the identified COI gene haplotypes. The evolutionary distances were computed using the Kimura two-parameter (K2P) model. Numbers between brackets in front of the nodes indicate bootstrap support (1,000 replicates). Boxes on the right indicate the best partition of species using ASAP, ABGD, mPTP and GMYC delimitation methods.

Figure 6. 

Pleopods 2 of males Metastenasellus as drawn in original descriptions (A) M. leleupi (scale 1) (B) M. camerounensis (scale 1) (C) M. dartivellei (scale 2) (D) M. powelli (scale 3) (E) M. congolensis (scale 1) (F) M. boutini (scale 2) (G) M. leysi (scale 3) (H) M. wikkiensis (scale 2) (I) M. tarrissei (scale 3).

Figure 7. 

Distribution map of Stenasellidae in Africa: Acanthastenasellus (A. forficuloides); Johanella (J. purpurea); Magniezia (Ma1: M. africana, Ma2: M. gardei, Ma3: M. guinensis, Ma4: M. laticarpa, Ma5: M. studiosorum); Metastenasellus (Me1: M. boutini, Me2: M. camerounensis, Me3: M. congolensis, Me4: M. dartvellei, Me5: M. leleupi, Me6: M. leysi, Me7: M. powelli, Me8: M. tarrissei, Me9: M. wikkiensis, Me10: Metastenasellus sp1, Me11: Metastenasellus sp2, Me12: Metastenasellus sp3, Me13: Metastenasellus sp. 4, Me14: Metastenasellus sp5); Parastenasellus (P. chappuisi); Stenasellus (St1: S. agiuranicus, St2: S. costai, St3: S. kenyensis, St4: S. migiurtinicus, St5: S. pardii, St6: S. ruffoi, St7: S. simonsi).

The new species M. boutini was collected in 7 of the 10 wells sampled in quarter PK21 of Douala (Table 1) city but was not found in the other 31 wells sampled in the same city. Isopod abundance in the wells was two-fold higher during the dry season than in the wet season.

The water chemistry of phreatic water in this part of the city was very acidic (pH = 4.2 ± 0.4), relatively warm, and well oxygenated with relatively low concentrations of calcium and magnesium (Table 2). We did not observe any statistically significant difference between the wells that harbored isopods and the wells without isopods (p-values > 0.092), whether in terms of physical traits (total depth, water depth) or physico-chemical parameters (Table 2). We also compared the seasonal variation of the stability of environmental conditions of the wells that harbored isopod vs. the wells without isopods, but no difference was observed (data not shown).

The known distribution of Stenasellidae in Africa is very patchy (most of the species are known only from their type localities). Genera belong to three main geographic groups (Fig. 7). One group located in the eastern part of the continent (Kenya and Somalia) include two genera and eight species: Acanthastenasellus forficuloides Chelazzi & Messana, 1985; Stenasellus agiuranicus Chelazzi & Messana, 1987; S. costai Lanza, Chelazzi & Messana, 1970 ; S. kenyensis Magniez, 1975; S. migiurtinicus Messana, Chelazzi & Lanza, 1974; S. pardii Lanza, 1966; S. ruffoi Messana, 1993; S. simonsi Messana, 1999. A second group is located in north-western Africa and includes three genera and 10 species. Most of them are located in western Africa: Parastenasellus chappuisi (Remy, 1938); Magniezia africana (Monod, 1945); M. gardei Magniez, 1978; M. guinensis (Braga, 1950), M. laticarpa (Birstein, 1972); M. studiosorum Sket, 1969. Only one species (Johannella purpurea Monod, 1924) of the genus Johanella is present in Algeria. The third group is only composed by the genus Metastenasellus and is widely distributed from the Democratic Republic of Congo to Algeria with 9 known species.

The first stenasellid isopods were found for the first time in France in 1896 and later described as Stenasellus virei Dolfus, 1897. This first species was followed by successive discoveries in southern and central Europe in the early 1900’s (Magniez 1999) together with the first African stenasellid, Johannella purpurea in Algeria (Monod 1924). More recent discoveries of new species in western, central, and eastern Africa have modified the taxonomy of the family in-depth, with the description of seven new genera since 1966 and especially the genus Metastenasellus (Magniez 1966). Magniez originally defined this genus as displaying well-developed pleonites 1 and 2, dactyli of pereopods 2–7 with one sternal spine, the male protopodite of pleopod 1 without a coupling hook and the male endopodite of pleopod 2 very voluminous and a helicoidal spermatic duct. A few years later, the diagnosis was slightly updated by changing the relative size of pleonites 1 and 2 compared to the length of pereonite 7 (Magniez 1979). In his original diagnosis, pleonites 1 and 2 reached at least 2/3 of pereonite 7. However, the discovery of M. wikkiensis Lincoln 1972 with reduced pleonites 1 and 2 (50% of pereonite 7) required an update of the diagnosis of the genus: the ratio of the length of pleonites 1 and 2 to the pereonite 7 has to be higher than 50%. Until the discovery of M. camerounensis in Cameroon (Zebaze Togouet et al. 2013), the ratio of 50% was restricted to M. wikkiensis. Our description of M. boutini with a similar ratio provides a third species of the genus with such a ratio and confirms the diagnostic validity of this criterium for the genus Metastenasellus.

Despite the geographical proximity of M. camerounensis and M. boutini, their morphology differs strongly. The two species have a relatively large size among Metastenasellus and a similar pleonites/pereonite 7 ratio, but the shape of pleopod 2 strongly differs. In M. boutini, the endopodite of pleopod 2 is evolved, with a distal part of the spermatic duct almost fully inside the second article of the endopodite. This specificity is considered as the ultimate evolution of pleopod 2 in the genus Metastenasellus (Magniez 1979), whereas the shape of the endopodite of M. camerounensis is much more primitive (50% of the spermatic duct is out of the second article) as for M. leleupi. By the shape of the pleopod 2, M. boutini is closer to M. wikkiensis found in Nigeria than to M. camerounensis. However, based on other characteristics such as the external lobes on the protopodite of pleopod 2, M. boutini is also close to the dwarf species M. leisi found in Algeria.

As suggested by previous studies, the second male pleopod exhibits several significant differences among Metastenasellus species (Magniez 1991; Zebaze Togouet et al. 2013). Among the many characteristics of pleopod 2, species can be separated into two main groups according to the shape of article 2 of the endopodite. The Metastenasellus leleupi group is characterized by a distal part of the spermatic duct clearly protruding out the second article of the endopodite (i.e a primitive characteristics sensu Magniez 1979); the group is composed of 5 species (M. leleupi, M. camerounensis, M. dartivellei, M. powelli, M. congolensis). The second group, the M. tarrissei group is composed of more evolved species whose distal part of the spermatic ducts is almost or fully inside the second article of the endopodite (M. boutini, M. tarrissei, M. wikkiensis, and M. leysi). However, the differences between these two taxonomic groups are not sufficient to distribute these species into two genera. Firstly, the morphological characteristics of all species fit the diagnosis of the genus for all traits (Magniez 1979), and secondly the difference among species are not dichotomous but follow a continuous gradient from M. leleupi to M. tarrissei.

The stygobitic family Stenasellidae is widely distributed in southwestern Europe, the Middle East, Asia, and even Central America (Magniez 1999; Lewis and Sawicki 2016). In Africa, stenasellid isopods can colonize a wide range of environmental niches under very different climate conditions ranging from dry Saharan climate to wet equatorial climate (Magniez, 1999). For instance, the family is known to be tolerant to a very wide range of pH values (from 3.4 for M. boutini in this study to 8 for Acanthastenasellus in Somalia) as well as a wide range of altitudes (from 0 to 1300 m a.s.l. for M. leysi in Algeria) and temperatures (more than 34 °C for S. rufoi in Kenya). They have large pleopods 3, 4, and 5 (gills) and their red/pink color indicates a high amount of haemolymph which likely enables them to withstand poor oxygen concentrations, even if this point has not been studied (Magniez 1999). However, and despite their wide environmental tolerance, there is no mention of stenasellids in brackish water and their known localities do not exceed a salinity of 3 g.L^-1for S. ruffoi (Messana, 1993).

Stenasellids can also colonize all kinds of groundwaters (karst, interstitial and phreatic waters) (Magniez 1999). The wide “tethyan” distribution of stenasellids suggests the presence of an ancestor at least during the Upper Cretaceous. This hypothesis is well supported by the molecular phylogeny of Asellota (Morvan et al. 2013) showing that stenasellids were already present on Pangaea during the late Paleozoic (≅ 250 MYA), while African and Nearctic stenasellids were geographically separated. The lack of data about the distribution of stenasellids in Africa does not allow us to draw a clear conclusion about their biogeography in Africa.

This identification key concerns males of the nine currently known species of the genus Metastenasellus: M. leleupi (Chappuis, 1951); M. congolensis (Chappuis, 1951); M. dartivellei (Chappuis, 1952); M. wikkiensis Lincoln, 1972; M. powelli Magniez, 1979; M. tarrissei Magniez, 1979; M. leysi Magniez, 1986; M. camerounensis Zebaze Togouet, Boulanouar, Njiné & Boutin, 2013, M. boutini Pountougnigni, Piscart & Zebaze (present study).

Like the key proposed by Zebaze Togouet et al. (2013), our key is largely based on the second male pleopod. However, the intermediate size of M. boutini did not allow us to just update the key proposed by Zebaze Togouet et al. (2013). As a consequence, the new key was largely rebuilt, as follows: