Taxonomic revision of the endemic Cameroonian freshwater crab genus Louisea Cumberlidge, 1994 (Crustacea, Decapoda, Brachyura, Potamonautidae), with descriptions of two new species from Nkongsamba and Yabassi

Abstract The taxonomy of the freshwater crab genus Louisea Cumberlidge, 1994, is reviewed based on type material and newly obtained specimens from three different localities in southwestern Cameroon. The genus is endemic to Cameroon and previously included two species: L.edeaensis (Bott, 1969) (type species) from Lake Ossa wetland complex (altitudes below 400 m asl) and L.balssi (Bott, 1959) from Kumba and Mt. Manengouba (altitudes above 1300 m asl). Here two new species of Louisea are described based on morphological and/or genetic data: L.nkongsambasp. nov. from the Nlonako Ecological Reserve (1000–1400 m asl) in the sub-montane zone and L.yabassisp. nov. from Yabassi in the lowlands. A redescription and amended diagnostic features of L.edeaensis and L.balssi are provided, and the genus diagnosis is updated to accommodate all four species. An identification key is also provided for the species of Louisea. A tree of phylogenetic relationships based on three mtDNA loci (COI, 12S rRNA, and 16S rRNA) supports the taxonomic revision, and indicates speciation of Louisea species along an altitudinal gradient, but further phylogenetic analyses are needed to understand whether this can lend support to the hypothesis that there is a montane centre of speciation along the Cameroon Volcanic Line. The phylogenetic tree also shows that Buea Cumberlidge, Mvogo Ndongo, Clark & Daniels, 2019 and Potamonemus Cumberlidge & Clark, 1992 are sister genera that may be derived from the Louisea lineage.


Introduction
The freshwater crab genus Louisea Cumberlidge, 1994, was established by Cumberlidge (1994) to accommodate two species from Cameroon.The first of these, L. edeaensis (Bott, 1969), was originally described by Bott (1969) as Globonautes macropus edeaensis Bott, 1969 from a single male specimen from Edea, Cameroon collected in 1910.The second species is L. balssi (Bott, 1959), which was described by Bott (1959) as Globonautes balssi based on specimens from Kumba, Cameroon collected between 1900 and 1910.This taxon was later treated by Bott (1970) as the subspecies Globonautes macropus balssi (Bott, 1970).Cumberlidge (1999) found that the genus Globonautes Bott, 1959, is endemic to the Upper Guinea Forests from Liberia to Guinea, and that Louisea is endemic to the Lower Guinea Forests in southwestern Cameroon.Both L. edeaensis and L. balssi were assessed as endangered (EN) under the International Union for Conservation of Nature (IUCN) Red List protocols, and it was thought at that time that both these species might even be extinct (IUCN 2003, Cumberlidge 2008a, 2008b, Cumberlidge et al. 2009).However, our recent biotic surveys in Cameroon (in August 2015 and May 2017) have led to the rediscovery of L. edeaensis from lowland forests below 400 m asl (Bedimet Island of the Lake Ossa wetland complex), and of L. balssi from high altitude forests above 1300 m asl (Mt.Manengouba) (Mvogo Ndongo et al. 2017aNdongo et al. , 2018)).The molecular data from the fresh specimens established the validity of the genus Louisea, and enabled the resolution of longstanding questions surrounding the phylogenetic relationships of the Louisea that was definitively assigned to the subfamily Potamonautinae Bott, 1970(Mvogo Ndongo et al. 2017c).
The rediscoveries of L. edeaensis and L. balssi prompted renewed extensive systematic surveys of the lowland and upland zones in the tropical rainforests of southwestern Cameroon in 2017 and 2018, which resulted in the recognition of a new species of Louisea from the submontane zone of Mt.Nlonako (1000-1400 m asl).This new species is described here based on morphological and genetic data.A second new species of Louisea is also recognised from Yabassi in the lowlands of southwestern Cameroon based on the re-examined specimens from the Museum für Naturkunde, Berlin, Germany (ZMB) that had previously been identified as belonging to Louisea (Cumberlidge 1994, 1999, Mvogo Ndongo et al. 2017a).The genus Louisea is revised here to include four species, L. edeaensis is redescribed, and the diagnosis of L. balssi is amended.The taxonomic revision of Louisea is based on a unique combination of characters of the carapace, thoracic sternum, chelipeds, and male first gonopods.It is also supported by original data on the phylogenetic relationships of Louisea and the other species of freshwater crabs found during our surveys of the rainforest ecosystems of southwestern Cameroon.The present molecular study used three mitochondrial genes (COI, 12S rRNA, and 16S rRNA) but included only three of the four species of Louisea (because no fresh specimens of the new species from Yabassi could be collected).Also included in the phylogenetic tree and species of the two other genera found in southwestern Cameroon: Buea Cumberlidge, Mvogo Ndongo, Clark &Daniels, 2019 andPotamonemus Cumberlidge &Clark, 1992.

Sampling
A series of field surveys of freshwater decapods undertaken in southwestern Cameroon from 2015 to 2018 focused on biodiversity hotspots that had been previously identified in 2011 by Conservation International for other freshwater taxa (Fig. 1).Crabs were hand-caught from puddles near small permanent streams, from under fallen leaves, and from their burrows sited close to water bodies.Specimens of Louisea sp.(ZMB Crust.21575) collected from Yabassi, Cameroon between 1900 and 1910 held in the Museum für Naturkunde were re-examined here.

Morphological analyses
All measurements (in mm) were taken with digital callipers.The terminology used follows Cumberlidge (1999), and the classification follows Ng et al. (2008).Characters of the gonopods, carapace, thoracic sternum, chelipeds, third maxillipeds, and mandibles were examined in detail, and photographs were taken using a Leica microscope (model Z16A POA), and LAS V4 and Helicon Focus 6.7.1 software.Post processing of the images was undertaken using Adobe Photoshop CC5.The type specimens of the two new species and the freshly collected specimens of Louisea species are deposited in the Museum für Naturkunde, Berlin, Germany (ZMB).Other material is deposited in the Institute of Fisheries and Aquatic Sciences, University of Douala at Yabassi (IFAS), the Senckenberg Museum, Frankfurt, Germany (SMF), the Zoological Institute Museum, Hamburg, Germany (ZIM), the Naturhistorisches Museum Wien, Austria (NHMW), and the Zoologische Sammlung des Bayerischen Staates, München, Germany (ZSBS).

Molecular analysis
Genomic DNA was extracted from a tissue sample of up to 25 mg cut from the pereiopod muscle of 70% ethanol-preserved specimens using the Qiagen DNeasy Blood & Tissue kit.
PCR products were sent to Macrogen Europe for purification and cycle sequencing of both strands of each gene.The sequences obtained were proofread manually using Chromas and aligned with Bioedit.Results from these genes were concatenated into a single alignment that was then converted into a Nexus file with FaBox (Villesen 2007).The best evolutionary model was determined with jModeltest v.2.1.7 (Darriba et al. 2012) based on the Akaike information criterion (Posada and Buckley 2004) and resulted in the GTR+I+G (COI), GTR+G (16S) and HKY+G (12S) models.The phylogenetic reconstruction was conducted with Maximum Likelihood (ML) using the software RAxML (Stamatakis 2006) under GTR + (I) + G model of sequence evolution.Bayesian Inference (BI) was performed to infer phylogeny by using MrBayes v. 3.2.2(Huelsenbeck and Ronquist 2001).The MCMC was run with four independent chains for 10,000,000 generations, samplefreq = 500, and burnin = 10,001.Analyses were conducted separately to test for topology congruence.The trees were drawn to scale, with branch lengths measured as the number of substitutions.All sequences generated for this study have been uploaded to GenBank (Table 1).Globonautes Bott 1959: 995, pl. 1, figs 1-6;1969: 359;1970: 23. Louisea Cumberlidge 1994: 123;1999: 226;Ng et al. 2008: 169 (list).
Remarks.Cumberlidge (1994Cumberlidge ( , 1999) ) provided the diagnostic characters of Louisea based mainly on the male holotype of L. edeaensis, because L. balssi was only known then from a juvenile male.The revision of the diagnostic characters for this genus is based on our examinations of adult males of all four species included here (L.edeaensis, L. balssi, L. nkongsamba sp.nov., and L. yabassi sp.nov.).The amended character descriptions are also used to compare Louisea with other potamonautid genera.
The bilobed terminal segment of the mandibular palp of Louisea is unusual, and it sets this genus apart from most genera in the Potamonautinae that typically possess a simple mandibular palp (i.e., with no additional anterior lobe) (Cumberlidge 1999;Cumberlidge et al. 1999;Cumberlidge and Reed 2003).This is true for Erimetopus Rathbun, 1894 and Platythelphusa A. Milne-Edwards, 1887, and all but one species of Potamonautes MacLeay, 1838 [P.brincki (Bott, 1960) being the exception], most species of Sudanonautes Bott, 1955[except for S. floweri (de Man, 1901) and S. orthostylis Bott, 1955], and most species of Liberonautes Bott, 1955(Cumberlidge 1999).It should be noted that in those potamonautine species without a simple mandibular palp, the anterior lobe is little more than a hard ledge at the junction between the segments, rather than a true lobe shape (Cumberlidge 1999).The terminal segment of the mandibular palp of Louisea is distinctly bilobed (with an anterior lobe 0.6 × the terminal segment length) and is superficially similar to the mandibular palps of Afrithelphusa Bott, 1969 and Globonautes (the two West African genera assigned to the Deckeniinae Ortmann, 1897, but the mandibular palp of the each of the latter two genera has a larger anterior lobe that is subequal to the posterior lobe (Fig. 15a-d; Cumberlidge 1999: fig.48A-C).The lack of a flagellum on the exopod of the third maxilliped in Louisea is rarely seen in other species of the Potamonautinae, and most of the members of this subfamily typically possess a long flagellum on the third maxilliped exopod (Cumberlidge 1999).The exceptions to this are the species of Buea and Potamonemus, and Liberonautes grandbassa Cumberlidge, 1999and L. lugbe Cumberlidge, 1999(Cumberlidge and Clark 1992;Cumberlidge 1993Cumberlidge , 1999;;Cumberlidge et al. 2019).The lack of a flagellum on the exopod of the third maxilliped of Louisea is also shared with species of the Deckeniinae (Afrithelphusa, Globonautes, and Madagapotamon humberti Bott, 1965) (Cumberlidge 1999;Cumberlidge et al. 2008).Louisea can also be distinguished from the West African Deckeniinae genera Afrithelphusa and Globonautes by characters of the gonopods (G1 TA shape and G2 TA length), the presence or absence of an intermediate tooth between the exorbital and epibranchial teeth (Cumberlidge 1999), and by molecular evidence (Daniels et al. 2015;Mvogo Ndongo et al. 2017c).(Bott, 1969) Figs 2a, 3a, 4c, 5a, 6a, 7a, 8a, b, 9a, 10a, 11a, 12a, 13a, 14a, 15a Globonautes macropus edeaensis Bott 1969: 360;1970: 24, pl. 1, figs 3-5, pl. 26, fig. 8;Cumberlidge 1987Cumberlidge : 2215, table , table , 1907, coll. Haberer (NHMW 1877).See Table 2 for details of the material examined from Lake Ossa.
Remarks.The description and diagnosis of L. edeaensis by Cumberlidge (1994) was based on characters of specimens from Edea and Yabassi.Mvogo Ndongo et al. (2017a) updated these characters following the discovery of a large series of L. edeaensis from Bedimet Island in Lake Ossa in August 2015.These specimens included only one adult male that agreed well with the diagnostic characters of the holotype from Edea, and with other specimens from Yaounde and Yabassi.Nevertheless, the morphological variations raised by Cumberlidge (1994) that distinguished the specimens from Yabassi from those from Yaounde, Edea, and Lake Ossa still remained.The specimens of Lake Ossa examined in this study included five adult males whose morphological characters are consistent with those from Edea and Yaounde, but different from the specimens from Yabassi.This resulted in the present re-description of L. edeaensis.Differences between L. edeaensis and its congeners are given below under general remarks.Louisea balssi (Bott, 1959) Figs 2b, 3b, 4b, 5b, 6b, 7b, 8c, d, 9b, 10b, 11b, 12b, 13b, 14b, 15b Globonautes balssi Bott 1959: 999, fig. 7;Cumberlidge 1987Cumberlidge : 2210;;1994  Carl Rathke (SMF 5093, donated by ZIM K3506).Other material examined is given in Table 3.
Remarks.Specimens of L. balssi are known only from Kumba and Mt.Manengouba in southwestern Cameroon.The morphological features of L. balssi are mainly defined from the adult male specimens collected from Mt. Manengouba (Mvogo Ndongo et al. 2018).The only available specimens from Kumba are sub-adults whose gonopod, sternal, and cheliped characters are not fully developed, which makes them less useful for taxonomic studies (Cumberlidge 1999).Further systematic surveys at the type locality near Kumba are needed to resolve this taxonomic problem but current social issues in this part of Cameroon preclude such surveys.Distinctions between L. balssi and its congeners are given below under general remarks.4c, 7c).Vertical sulcus on carapace branchiostegal wall curving backward to meet anterolateral margin at epibranchial tooth (Fig. 6c).Mandibular palp bi-segmented; terminal segment (TS) bilobed, with large distinct anterior lobe 0.6 × terminal segment length (Fig. 15c).Third maxilliped ischium with distinct vertical groove (Fig. 14c).Episternal sulci s4/e4, s5/e5, s6/e6 faint or missing, s7/e7 complete (Fig. 5c).Major cheliped dactylus highly arched enclosing oval interspace when closed, with five large teeth (one small distal, two large medial, two small proximal) (Fig. 8e); propodus of major cheliped with two large proximal teeth, large medial tooth, small distal tooth (Fig. 8e); cheliped carpus inner margin with long, broad distal tooth, relatively narrow, subequal proximal tooth (Fig. 10c).G1 TA short (TA/SS 0.22), directed outwards at 45° angle to longitudinal axis of G1 SS, with distinct longitudinal groove proximally distinctly broad, abruptly narrow, slim and tube-like at distal two-thirds (Figs 11c, 12c).G1 SS tapering slightly from broad basal margin to relatively wide distal margin (0.5 × SS basal margin); dorsal face with broad dorsal membrane (maximum width 0.1 × SS length) at TA/SS junction (Fig. 11c).G2 TA long (TA/SS 0.44), flagellum-like, almost as long as G2 SS (Fig. 13c).Mature at CW 19 mm.
Male pleon triangular, sides not indented with small setae; telson (a7) rounded at distal margin (Fig. 5c).G1 TA short (TA/SS 0.22), directed outwards at 45° angle to longitudinal axis of G1 SS, with distinct longitudinal groove on ventral face, proximally distinctly broad, abruptly narrow, slim and tube-like at distal two-thirds (Figs 11c,12c).G1 SS tapering slightly from broad basal margin to relatively wide distal margin (0.5 × SS basal margin); ventral face of with raised triangular flap extending halfway across segment forming roof of chamber for G2, flap tapering diagonally from broad base to narrow point at SS/TA junction (Fig. 12c); dorsal face with broad dorsal membrane (maximum width 0.1 × SS length) at TA/SS junction (Fig. 11c).G2 TA long (TA/SS 0.44), flagellum-like, almost as long as G2 SS (Fig. 13c).Mature at CW 19 mm.
Color in life.Unknown.Type locality.Yabassi, littoral region of Cameroon.
Etymology.The new species is named for Yabassi in south-western Cameroon, where it was collected in 1909.The species epithet is a noun in apposition.
Habitat.Louisea yabassi sp.nov. is known only from Yabassi, a humid area of the coastal rain forest of southwestern Cameroon.It is possible that this species is also present in the Ebo forest near Yabassi, which is one of the largest remaining tracts of lowland and submontane rainforest in the area.
Color in life.Specimens of L. nkongsamba sp.nov.have a dark brown or green carapace and walking legs.
Etymology.The new species is named for Nkongsamba, the closest town to the type locality.The species epithet is a noun in apposition.
Habitat.L. nkongsamba sp.nov. is known only from Nlonako Wildlife Reserve, one of the threatened tropical rainforest habitats in the littoral region of Cameroon.
Remarks.L. nkongsamba sp.nov.possesses numerous characters that link it to L. edeaensis, L. balssi, and L. yabassi sp.nov.Differences between these species are discussed below under general remarks.

General remarks
The generic characters of Louisea proposed by Cumberlidge (1994Cumberlidge ( , 1999) ) were based on L. edeaensis and a subadult specimen of L. balssi.These characters are therefore reassessed here in the light of recently-rediscovered populations of L. edeaensis and L. balssi that included adult males of both species (Mvogo Ndongo et al. 2017a, 2018), and the two new species from Yabassi and Mt.Nlonako described here.All these four species are assigned to Louisea because they share the emended diagnostic characters for the genus presented here, and three of them at least form a monophyletic group (Fig. 16).The main characters that differentiate the four species are based on the cheliped dactylus; the inner margin teeth on the cheliped carpus; the intermediate tooth between exorbital and epibranchial teeth; the postfrontal crest; sternal segment S3; sternal sulcus S2/S3; the anterior lobe on the TS of the mandibular palp; and the G1 TA length.Some of these characters are shared by two of the four species.The differences between the four species of Louisea are given in Table 5.

Discussion
The highlands of southwestern Cameroon are part of the continental segment of the Cameroon Volcanic Line (CVL) that includes Mount Cameroon, Mt.Manengouba, Mt.Nlonako, Mt.Lefo, Mt.Oku, and the Ngaoundere Plateau (Burke 2001;Zimkus 2009).The CVL crosses a significant portion of the West African forest biodiversity hotspot, which is remarkably species rich and has a high rate of endemism (Stuart 1986;Lawson 1993;Bowden and Andrews 1994;Stattersfield et al. 1998;Myers et al. 2000;Lovett and Taplin 2004;Herrmann et al. 2005).Although the freshwater crab fauna of southwestern Cameroon is also species-rich and has a high number of endemic species (Cumberlidge et al. 2019), most parts of this part   (Fig. 15d) Medium (0.6× TS length) (Fig. 15c) Small (0.5× TS length) (Fig. 15b) Medium (0.6× TS length) (Fig. 15a) G1 TA length Short (0.22× G1 length) (Fig. 11d) Medium (0.27× G1 length) (Fig. 11c) Medium (0.28× G1 length) (Fig. 11b) Medium (0.29× G1 length) (Fig. 11a) of the country have remained largely unsurveyed for freshwater crabs until recently (Mvogo Ndongo et al. 2017a, 2017b, 2017c, 2018).Recent biotic surveys of the freshwater crab fauna in southwestern Cameroon coupled with the re-examination of museum specimens have led to the discovery or rediscovery of a number of taxa, raising the number of species from Cameroon to 17, and the number of genera to five (Cumberlidge et al. 2019).However, the two rediscovered species Louisea edeaensis and L. balssi and the two new species of Louisea described here are all from new localities, because attempts to find additional specimens of Louisea were not successful at Yabassi, Edea, Yaounde and Kumba, localities where they were collected between 1900 and 1910.It is possible that the populations of Louisea in these four locations have been extirpated because they were last found in 1910, and there has been considerable human population expansion and urban development in these areas since then.This is especially unfortunate because the specimens from Yabassi have a problematic taxonomic history, but the larger series of specimens of all species in this genus now available enables us to recognise the specimens from Yabassi as a new species, L. yabassi sp.nov.Our phylogenetic analyses (Fig. 16) based on 1801 base pairs of three mitochondrial genes (combined COI, 16S RNA, 12S RNA) found strong BI and ML support for the continued recognition of the genus Louisea with a well-supported clade that includes L. edeaensis, L. balssi, and L. nkongsamba sp.nov.We were not able to extract DNA from the available specimens of L. yabassi sp.nov., and this species was therefore not included in the tree.It has been suggested that the montane regions on the Cameroon Volcanic Line act as centres of speciation, as has been reported by Zimkus (2009) for puddle frogs from Mt. Oku.Our data indicate that the highland dwelling species L. balssi is the sister group to the two other species of Louisea found at lower altitudes, which indicates that speciation may have happened along an altitudinal gradient.However, further phylogenetic analyses are needed to establish whether this can lend support to the hypothesis that CVL is a montane centre of speciation.It is interesting to note that our phylogenetic tree also recognizes for the first time two distinct lineages within the genus Buea and one lineage within Potamonemus (Mvogo Ndongo et al. in prep).Buea and Potamonemus are sister genera and are apparently derived from the well-established diversified lineages of Louisea, but a phylogenetic work that includes all known genera assigned to the Potamonautinae is needed to test this hypothesis.

Figure 1 .
Figure 1.Collection localities of the four species of Louisea in Cameroon.

Figure 16 .
Figure 16.ML tree topology for the freshwater crab taxa from Cameroon included in this study derived from mtDNA sequences corresponding to three loci (partial 16S rRNA, COI and 12S rRNA genes).BI and ML statistical values (%) on the nodes indicate posterior probabilities and bootstrap support, respectively.
Size of anterior lobe on terminal segment (TS) of mandibular palp Large (0.8× TS length)

Table 1 .
Species of Louisea, Buea, Potamonemus and outgroups included in the molecular analyses.The newly-presented data are given in bold.

Table 4 .
Morphometric analysis and collection data of specimens (N = 27) of Louisea nkongsamba sp.nov.from Cameroon.All measurements are given in mm.

Table 5 .
Differences between the four species of the genus Louisea.