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Research Article
Phylogenetic relationships among the species of the Cameroonian endemic freshwater crab genus Louisea Cumberlidge, 1994 (Crustacea, Brachyura, Potamonautidae), with notes on intraspecific morphological variation within two threatened species
expand article infoPierre A. Mvogo Ndongo§, Thomas von Rintelen§, Paul F. Clark|, Adnan Shahdadi, Carine Rosine Tchietchui#, Neil Cumberlidge¤
‡ Université de Douala à Yabass, Douala-Bassa, Cameroon
§ Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, Berlin, Germany
| Department of Life Sciences, The Natural History Museum, London, United Kingdom
¶ University of Hormozgan, Bandar Abbas, Iran
# University of Douala, Douala, Cameroon
¤ Northern Michigan University, Marquette, United States of America
Open Access

Abstract

Louisea Cumberlidge, 1994 (Crustacea, Brachyura, Potamonautidae) currently includes four endemic Cameroonian freshwater crab species whose phylogenetic relationships were previously unresolved. In the present study, phylogenetic analyses are carried out involving three mtDNA loci (COI, 12S rRNA, and 16S rRNA). The COI locus revealed divergence times of 5.6 million years ago (myr) for when L. balssi (Bott, 1959) diverged from L. edeaensis (Bott, 1969); 4.1 myr for when L. edeaensis diverged from L. yabassi Mvogo Ndongo, von Rintelen & Cumberlidge, 2019; and 2.48 myr for when the later species diverged from L. nkongsamba Mvogo Ndongo, von Rintelen & Cumberlidge, 2019. Three genetic lineages were found within L. nkongsamba that are supported by uncorrected p-distances and the haplotype network. Morphological variation in some taxonomically important characters was found within both L. nkongsamba and L. yabassi. No correlation, however, was found between the morphotypes within these species and the uncovered genetic lineages. Recognition of species boundaries and of subpopulations of species will prove valuable when making informed conservation decisions as part of the development of species action plans for these rare and threatened freshwater crabs.

Keywords

Decapoda, morphotypes, Nkongsamba, Potamoidea, species boundaries, Yabassi

Introduction

Louisea Cumberlidge, 1994 (Crustacea, Brachyura, Potamonautidae) is endemic to remote Cameroonian forested ecosystems and currently includes four freshwater crab species: L. balssi (Bott, 1959), L. edeaensis (Bott, 1969), L. nkongsamba Mvogo Ndongo, von Rintelen & Cumberlidge, 2019, and L. yabassi Mvogo Ndongo, von Rintelen & Cumberlidge, 2019. Louisea balssi and L. edeaensis have been revised recently based on new material collected in Cameroon (Mvogo Ndongo et al. 2017a, 2018, 2019), while L. nkongsamba and L. yabassi were recently discovered (Mvogo Ndongo et al. 2019). Other works on Cameroonian freshwater crabs have mainly focused on their taxonomy, phylogenetic relationships, or conservation (Cumberlidge 1999; Daniels et al. 2015; Mvogo Ndongo et al. 2017a, 2017b, 2017c, 2018, 2019, 2020, 2021a; Cumberlidge and Daniels 2022). This is the first study, however, that includes both morphological and molecular data from all known Louisea species. The present work also includes new collections of two Louisea species from the forested sites in southwestern Cameroon: L. yabassi from the Ebo Forest, and L. nkongsamba from the Nlonako Ecological Reserve (Mvogo Ndongo et al. 2019, 2021b). These populations are compared with those of L. balssi from Kumba and Mount Manengouba (Cumberlidge 1994, 1999; Mvogo Ndongo et al. 2017a, 2017c, 2018, 2019), and of L. edeaensis from Yaounde, Edea, and Lake Ossa (Cumberlidge 1994, 1999; Mvogo Ndongo et al. 2017a, 2017c, 2019).

The aim of the present work is to evaluate the phylogenetic relationships within Louisea and to estimate the genetic distance between the species using molecular data. Intraspecific variation of some important taxonomic characters within two newly discovered species is also assessed in order to better identify species boundaries within Louisea. Accurate species delimitation is necessary for understanding levels of biodiversity, and for adopting effective conservation and sustainable management strategies (Cornetti et al. 2015). The results from this study will be helpful in developing action plans aimed at the conservation of these rare, threatened, and endemic Cameroonian freshwater crab species.

Materials and methods

Sample collection

Four Louisea species were collected from four different locations in southwestern Cameroon between 2015 and 2021 (Fig. 1). The species were identified by following Cumberlidge (1994, 1999) and Mvogo Ndongo et al. (2019). Eight specimens of L. balssi were collected from 1,958 m a.s.l., Mount Manengouba; 30 specimens of L. edeaensis from 90 m a.s.l., Bedimet Island, Lake Ossa; 50 specimens of L. nkongsamba from 1000–1400 m a.s.l., Mount Nlonako; and 35 specimens of L. yabassi from up to 300 m a.s.l., the Ebo Forest near Yabassi. Specimens of L. nkongsamba and L. yabassi were studied to clarify intraspecific morphological variation within each species. Specimens were measured; their gender and life stage (juvenile, subadult, and adult) recorded; and their habitat preferences noted. Most of the crabs were released into their natural habitat after recording all relevant morphological data. Only a few whole adult specimens (males and females), as well as one of the walking legs was removed from each of the other selected specimens were preserved in ethanol for further morphological descriptions and molecular analyses. The newly collected specimens were deposited either in the Museum für Naturkunde, Berlin, Germany (ZMB), or in the Unity of Taxonomy, Production and Sustainable Management of Aquatic Animals, Department of Management of Aquatic Ecosystems, Institut des Sciences Halieutiques, University of Douala, Cameroon (LABO-PASMAT).

Figure 1. 

Map of Cameroon showing collection sites of Louisea species. Louisea nkongsamba: type locality (green triangle), new localities (green circles); Louisea yabassi: type locality (purple hexagon), new localities (purple circles).

Morphological analyses

Descriptive morphometrics of L. edeaensis and L. balssi specimens are given in Mvogo Ndongo et al. (2019: tables 2 and 3, respectively). Measurements (in mm) of the carapace of all the specimens were made with digital callipers. Characters of the carapace, thoracic sternum, chelipeds, and mandibles were examined in detail. The terminology used follows Cumberlidge (1999), and the classification by Cumberlidge and Daniels (2022). Images of the body parts were taken using a Leica microscope (model Z16A POA), and the LAS v. 4 and Helicon Focus v. 6.7.1 software. Post processing was undertaken using Adobe Photoshop CC5. Specimens were sorted according to their stage of development into juveniles, subadults, and adults. Furthermore, the maturity of adults was deciphered by identifying specimens that had undergone the pubertal moult from subadult to adult. The pubertal moult was determined by examining the degree of development of the pleon of a series of juvenile, subadult and adult females. The pleon of juvenile females is undeveloped and resembles the slim pleon of juvenile males; the pleon of subadults is significantly widened and partially covers the thoracic sternum. In comparison, the pleon of adult females is conspicuously enlarged and rounded such that its lateral margins overlap the coxae of the pereiopods, and the telson covers thoracic sternites 1 and 2. The lower limit of the range for the pubertal moult was judged as the CW of the largest non-adult female, while the upper limit of the pubertal moult was the CW of the smallest adult female.

Molecular analyses

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 following the manufacturer’s instructions. Polymerase chain reaction (PCR) was used to amplify three mitochondrial gene fragments: a ~638 bp region of the 16S ribosomal RNA gene (16S rRNA) using primers 16L29 and 16HLeu (Schubart 2009); a ~594 bp region of the 12S ribosomal RNA gene (12S rRNA) using primers 12L4 and 12H2 (Schubart et al. 2006); and a 648 bp region of the protein-coding mitochondrial gene, cytochrome oxidase subunit I gene (COI) using primers LCO-1490 and HCO-2198 (Folmer et al. 1994). PCR was performed in 25 μl volumes containing 1× Taq buffer, 1.5 mM MgCl2, 200 μM each dNTP, 1 U Taq polymerase, ~50–100 mg DNA and ddH2O up to volume. After an initial denaturation step of 4 min at 94 °C, cycling conditions were 35 cycles at 94 °C for 30 s, 45 °C for 60 s, and 72 °C for 90 s, with a final elongation step of 5 min at 72 °C. The same primers were used in PCR and sequencing. 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 Lite (v. 2.1.1) (Technelysium Pty Ltd, Queensland, Australia) and aligned with ClustalW (Thompson et al. 1994) implemented in BioEdit 7.0.5 (Hall 1999). New sequences were submitted to the National Center for Biotechnology Information and are available from GenBank under the accession numbers in Table 1. Results from these genes were concatenated into a single alignment, which was then converted into a Nexus file with FaBox (Villesen 2007).

Table 1.

Details of mtDNA markers used in the present study for Louisea species and outgroup species. Nl = Nlonako; Here = sequence available in the present study; * = Mvogo Ndongo et al. 2019; ** = Mvogo Ndongo et al. 2017c.

Species and sample number Locality in Cameroon Population number Morphotypes (see Tables 3, 4) Museum/extraction number GenBank accession number
COI 12S rRNA 16S rRNA
L. nkongsamba (1) Nlonako, Engugue1382 Population 1 Nl Morphotype 1 ZMB-X21 OP122926 OP133321 OP133281
L. nkongsamba (2) Nlonako, NgaltongueS1 Population 1 Nl Morphotype 1 ZMB-X26 OP122931 OP133326 OP133286
L. nkongsamba (3) Nlonako, NgaltongueS1 Population 1 Nl Morphotype 1 ZMB-X27 OP122932 OP133327 OP133287
L. nkongsamba (4) Nlonako, NgaltongueS1 Population 1 Nl Morphotype 1 ZMB-X28 OP122933 OP133328 OP133288
L. nkongsamba (5) Nlonako, NgaltongueS1 Population 1 Nl Morphotype 1 ZMB-X29 OP122934 OP133329 OP133289
L. nkongsamba (6) Nlonako Engugue1462 Population 1 Nl Morphotype 1 ZMB-X31 OP122936 OP133331 OP133291
L. nkongsamba (7) Nlonako, NgaltongueS2 Population 1 Nl Morphotype 1 ZMB-X36 OP122941 OP133336 OP133296
L. nkongsamba (8) Nlonako, NgaltongueS2 Population 1 Nl Morphotype 1 ZMB-X37 OP122942 OP133337 OP133297
L. nkongsamba (9) Nlonako, NgaltongueS2 Population 1 Nl Morphotype 1 ZMB-X38 OP122943 OP133338 OP133298
L. nkongsamba (10) Nlonako, NgaltongueS2 Population 1 Nl Morphotype 1 ZMB-X39 OP122944 OP133339 OP133299
L. nkongsamba (11) Nlonako, Eyimba Population 1 Nl Morphotype 1 ZMB-X41 OP122946 OP133341 OP133301
L. nkongsamba (12) Nlonako, Nguengue Population 1 Nl Morphotype 1 ZMB-X46 OP122951 OP133346 OP133306
L. nkongsamba (13) Nlonako, Nguengue Population 1 Nl Morphotype 1 ZMB-X47 OP122952 OP133347 OP133307
L. nkongsamba (14) Nlonako, Nguengue Population 1 Nl Morphotype 1 ZMB-X48 OP122953 OP133348 OP133308
L. nkongsamba (15) Nlonako, Engugue1382 Population 2 Nl Morphotype 1 ZMB-X22 OP122927 OP133322 OP133282
L. nkongsamba (16) Nlonako, Engugue1382 Population 2 Nl Morphotype 1 ZMB-X23 OP122928 OP133323 OP133283
L. nkongsamba (17) Nlonako, Engugue1382 Population 2 Nl Morphotype 1 ZMB-X24 OP122929 OP133324 OP133284
L. nkongsamba (18) Nlonako, NgaltongueS1 Population 2 Nl Morphotype 1 ZMB-X30 OP122935 OP133330 OP133290
L. nkongsamba (19) Nlonako Engugue1462 Population 2 Nl Morphotype 2 ZMB-X32 OP122937 OP133332 OP133292
L. nkongsamba (20) Nlonako Engugue1462 Population 2 Nl Morphotype 2 ZMB-X33 OP122938 OP133333 OP133293
L. nkongsamba (21) Nlonako Engugue1462 Population 2 Nl Morphotype 2 ZMB-X34 OP122939 OP133334 OP133294
L. nkongsamba (22) Nlonako, Eyimba Population 2 Nl Morphotype 1 ZMB-X42 OP122947 OP133342 OP133302
L. nkongsamba (23) Nlonako, Eyimba Population 2 Nl Morphotype 1 ZMB-X43 OP122948 OP133343 OP133303
L. nkongsamba (24) Nlonako, Eyimba Population 2 Nl Morphotype 1 ZMB-X44 OP122949 OP133344 OP133304
L. nkongsamba (25) Nlonako, Nguengue Population 2 Nl Morphotype 1 ZMB-X49 OP122954 OP133349 OP133309
L. nkongsamba (26) Nlonako, Nguengue Population 2 Nl Morphotype 1 ZMB-X50 OP122955 OP133350 OP133310
L. nkongsamba (27) Nlonako, Engugue1382 Population 3 Nl Morphotype 1 ZMB-X25 OP122930 OP133325 OP133285
L. nkongsamba (28) Nlonako Engugue1462 Population 3 Nl Morphotype 1 ZMB-X35 OP122940 OP133335 OP133295
L. nkongsamba (29) Nlonako, NgaltongueS2 Population 3 Nl Morphotype 1 ZMB-X40 OP122945 OP133340 OP133300
L. nkongsamba (30) Nlonako, Eyimba Population 3 Nl Morphotype 1 ZMB-X45 OP122950 OP133345 OP133305
L. yabassi (31) Eboforest Stream no. 1 Population 1 Ebo Morphotype 1 ZMB-X11 OP122956 OP133351 OP133311
L. yabassi (32) Eboforest Stream no. 1 Population 1 Ebo Morphotype 1 ZMB-X12 OP122957 OP133352 OP133312
L. yabassi (33) Eboforest Stream no. 1 Population 1 Ebo Morphotype 1 ZMB-X13 OP122958 OP133353 OP133313
L. yabassi (34) Eboforest Stream no. 1 Population 1 Ebo Morphotype 1 ZMB-X14 OP122959 OP133354 OP133314
L. yabassi (35) Eboforest Stream no. 1 Population 1 Ebo Morphotype 1 ZMB-X15 OP122960 OP133355 OP133315
L. yabassi (36) Eboforest Stream no. 2 Population 2 Ebo Morphotype 2 ZMB-X16 OP122961 OP133356 OP133316
L. yabassi (37) Eboforest Stream no. 2 Population 2 Ebo Morphotype 2 ZMB-X17 OP122962 OP133357 OP133317
L. yabassi (38) Eboforest Stream no. 2 Population 2 Ebo Morphotype 2 ZMB-X18 OP122963 OP133358 OP133318
L. yabassi (39) Eboforest Stream no. 2 Population 2 Ebo Morphotype 2 ZMB-X19 OP122964 OP133359 OP133319
L. yabassi (40) Eboforest Stream no. 2 Population 2 Ebo Morphotype 2 ZMB-X20 OP122965 OP133360 OP133320
L. edeaensis Lake Ossa, Bedimet Island Population 1 ZMB Crust 30335 MN188068.1* MN217395*
L. edeaensis Lake Ossa, Bedimet Island Population 1 T351-30 KY964474.1** KY964479** KY964472**
L. edeaensis Lake Ossa, Bedimet Island Population 1 ZMB_Crust 26930 KY964473.1** KY964478**
L. balssi Manengouba, stream Population 1 ZMB Crust 30319 MN188071.1* MN217385* MN217392*
L. balssi Manengouba, stream Population 1 ZMB Crust.29628 MN188070.1* MN217384* MN217391*
Potamonemus man Bakossi National Park Population 1 ZMB Crust 30327 MN188067.1* MN217390* MN217398*
Buea mundemba Korup National Park Population 1 ZMB Crust 30321 MN188069.1* MN217388* MN217396*

Phylogeographic investigations

The COI mitochondrial gene employed here is relatively variable and is commonly used for population genetics, and more recently also for faunal species identification using the barcoding approach (Hebert et al. 2003). This was useful for the examination of the population structure of L. nkongsamba, which provides evidence for genetic substructure among the sampling sites in Nlonako Ecological Reserve. These data are critical for the investigation of the historical connectivity among populations of Louisea species and are useful for the implementation of the future management of genetic diversity.

Maximum parsimony genotype networks (Templeton et al. 1992) were built with the software PopArt (Leigh and Bryant 2015) in order to graphically depict the genetic distances between mitochondrial genotypes. Haplotype and nucleotide diversities were used to compare genetic diversities among the sampling sites in terms of the number of haplotypes and the genetic distances of these haplotypes. Phylogeographic investigations have been successfully used by several researchers to determine connectivity among populations of other endemic crab species, e.g., Sesarma fossarum Schubart, Reimer, Diesel & Türkay, 1997, from the Cockpit Country, Jamaica (see Stemmer and Schubart 2016).

Phylogenetic investigations

The mitochondrial genes (COI, 12S rRNA, 16S rRNA) were used to identify the species boundaries, to examine the evolutionary origins and the relationships within Louisea species, and to determine whether morphological and ecological similarities between species are based on convergence or common ancestry. Here two methods of phylogenetic inference were applied to the data set: maximum likelihood (ML) using the software PAUP*, and Bayesian inference (BI) as implemented in MrBayes (v. 3.3; Huelsenbeck and Ronquist 2001) (see Mvogo Ndongo et al. 2017b, 2017c; Fratini et al. 2005). 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 rRNA) and HKY+G (12S rRNA) models. ML tree was obtained for each alignment with 1000 bootstrap pseudoreplicates. BI was performed to infer phylogeny by using MrBayes v. 3.2.2 (Huelsenbeck and Ronquist 2001). The Markov Chain Monte Carlo 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.

A total of 138 DNA sequences were obtained, 46 sequences each of COI, 16S rRNA, and 12S rRNA (Table 1). ML and BI trees were constructed for individual gene. The relative tree presented here for ML topology has been reconstructed from the concatenation of the three partial loci (COI, 16S rRNA, and 12S rRNA) into a single alignment, which was then converted into a Nexus file with FaBox. This tree includes L. balssi, L. edeaensis, L. nkongsamba, and L. yabassi as the in-group, and Potamonemus man Mvogo Ndongo, von Rintelen & Cumberlidge, 2021a and Buea mundemba Mvogo Ndongo, von Rintelen & Cumberlidge in Mvogo Ndongo, von Rintelen, Tomedi-Tabi & Cumberlidge, 2020 as the out-group species.

To estimate clade divergence times based on the COI gene, a Bayesian analysis with the software BEAST v. 2.6.2 (Bouckaert et al. 2019) was conducted using a strict clock model (Yule Model) with a rate of evolution for the COI of 2.33% per million years (my) (10% SD) (following Schubart et al. 1998). Markov chains for 10 million generations were undertaken, sampling every 1000th iteration and discarding the first 25% as burn-in. Overall, 7500 trees were obtained, and these trees were used to calculate the maximum clade credibility tree in TreeAnnotator v. 1.6.1 (part of the BEAST package). The uncorrected p-distances (%) was calculated in MEGA 7 (Kumar et al. 2016).

Abbreviations used

a.s.l. above sea level;

CW carapace width measured at widest point;

CL carapace length measured along medial line from anterior to posterior margin;

CH carapace height measured at maximum height of cephalothorax;

FW front width measured along anterior frontal margin between inner angles of orbits;

myr million years ago;

PAMN Pierre A. Mvogo Ndongo;

S2/3 male sternal sulcus between thoracic sternites 2 and 3;

S3/4 male sternal sulcus between thoracic sternites 3 and 4.

Results

Morphological analyses

Morphometric measurements of L. yabassi and L. nkongsamba populations are provided in Table 2. The adult size range of L. yabassi, based on male and female specimens from the two populations, was determined to be between CW 16.5 and CW 24.0 mm. Subadults of L. yabassi ranged from CW 11.0 mm to CW 15.5 mm, whereas juveniles of this species were CW 10.0 mm or less. The adult size range of L. nkongsamba, based on male and female specimens from four of the six sites, was between CW 15.8 mm and CW 20.0 mm. Subadults of L. nkongsamba ranged from CW 11.5 mm to CW 14.4 mm (two populations, PAMN 02.12.19 and PAMN 10.12.19, consisted entirely of subadults), whereas juveniles of this species measured CW 10.0 mm or less. No major differences were found between the carapace proportions (CW/FW, CL/FW, and CH/FW) of any of the populations of these two species, and these proportions were virtually identical in all cases (Table 2). The difference between the adult size range of L. yabassi and L. nkongsamba is minor, with the former species growing up to CW 24 mm and the latter species reaching only CW 20 mm.

Table 2.

Morphometric and collection data of specimens of Louisea yabassi from Ebo Forest, Cameroon, and L. nkongsamba from Nlonako Ecological Reserve, Cameroon. Ad: adult; Sa: subadult, M: male; F: female.

Species CW/FW mean (n) CL/FW mean (n) CH/FW mean (n) Size range (CW in mm) Museum number Locality Geographic coordinates Altitude (m a.s.l.)
L. yabassi 2.9 (19) 2.1 (19) 1.3 (19) Ad M 16.4–20.2 LaboPasmat X100 Ebo Forest, Stream NO. 1 04°25'01.7"N, 010°12'00.8"E 162
L. yabassi Ad F 12.0–24.1 ZMB Crust 33829 Ebo Forest, Stream NO. 1 04°25'01.7"N, 10°12'00.8"E 162
L. yabassi 2.9 (16) 2.1 (16) 1.3 (16) Ad M 16.6–21.3 LaboPasmat X101 Ebo Forest, Stream NO. 2 04°24'59.3"N, 010°12'07.7"E 254
L. yabassi Ad F 17.4–22.5 ZMB Crust.33775 Ebo Forest, Stream NO. 2 04°24'59.3"N, 010°12'07.7"E 254
L. nkongsamba 2.9 (8) 2.1 (8) 1.3 (8) Ad M 15.8–20.0 LaboPasmat X102 Nlonako, Nguengue 04°54'44.8"N, 009°58'50.0"E 1176
L. nkongsamba 2.9 (5) 2.1 (5) 1.3 (5) Ad M 12.8–18.5 LaboPasmat X102Y Nlonako, NgaltongueS2 04°55'20.4"N, 009°57'31.0"E 1180
L. nkongsamba 2.9 (12) 2.1 (12) 1.3 (12) Ad M 13.8–17.4 LaboPasmat X103 Nlonako, NgaltongueS1 04°55'20.4"N, 009°57'42.6"E 1180
L. nkongsamba 2.9 (10) 2.1 (10) 1.3 (10) Sa M 11.7–11.8 ZMB Crust.33789 Nlonako, Engugue1382 04°54'21.6"N, 009°58'20.6"E 1382
L. nkongsamba 2.9 (11) 2.1 (11) 1.3 (11) Sa M 11.5–14.4 LaboPasmat X104 Nlonako, Eyimba 04°53'30.7"N, 009°59'05.1"E 1194
L. nkongsamba 2.9 (6) 2.1 (6) 1.3 (6) Ad 12 LaboPasmat X104Y Nlonako, Engugue1462 04°54'21.9"N, 009°58'22.4"E 1462
L. nkongsamba Ad F 14–15 LaboPasmat X105 Nlonako, Engugue1462 04°54'21.9"N, 009°58'22.4"E 1462
L. nkongsamba Sa 6.60 LaboPasmat X105Y Nlonako, Engugue1462 04°54'21.9"N, 009°58'22.4"E 1462
L. edeaensis 3.0 (21) 2.5 (21) 1.4 (21) Ad M 14.1–17.5 See Mvogo Ndongo et al. 2019: 143, table 2 Lake Ossa 03°48'56.1"N, 010°03'18.5"E 90
L. edeaensis Ad F 13.0–19.9 See Mvogo Ndongo et al. 2019: 143, table 2 Lake Ossa 03°48'56.1"N, 010°03'18.5"E 90
L. balssi 2.9 (8) 2.1 (8) 1.2 (8) Ad M 13.3–16.2 See Mvogo Ndongo et al. 2019: 147, table 3 Manengouba 05°01'56.9"N, 009°49'37.8"E 1958
L. balssi Ad F 13.3–14.8 See Mvogo Ndongo et al. 2019: 147, table 3 Manengouba 05°01'56.9"N, 009°49'37.8"E 1958

Differences in certain morphological characters of the specimens of L. yabassi from two populations in the Ebo Forest are noteworthy (Table 3). Like L. yabassi, L. nkongsamba also showed differences in several morphological characters among the specimens from six sites, which are organised here into morphotype 1 (Nlonako Enguegue NO. 1_1462) and morphotype 2 (Nlonako Eyimba, Ngaltongue, Enguegue NO. 2_1382 m, Nguegue) (Table 4). These morphological differences between the two populations/morphotypes of L. yabassi and L. nkongsamba are also illustrated (Figs 2, 3). Despite those morphological differences, there is no genetic support for recognising these differences as indicating different genetic lineages that would warrant formal taxonomic recognition (Figs 46).

Table 3.

Comparison of selected morphological characters between two populations (morphotypes) of Louisea yabassi from Ebo Forest, Cameroon.

Character Population no. 1 (morphotype 1) Population no. 2 (morphotype 2)
Epibranchial tooth reduced to granule (Fig. 2A, C) small (Fig. 2B, D)
Intermediate tooth between exorbital & epibranchial teeth distinct, but small (Fig. 2A, C) relatively large, triangular (Fig. 2B, D)
Major cheliped dactylus slim, gently arched (Fig. 3F) slim, almost straight (Fig. 3H)
Cheliped carpus inner margin teeth both distal and proximal teeth large, positioned some distance from each other (Fig. 3G) distal tooth larger than proximal tooth, positioned relatively closer to each other (Fig. 3C)
Mandible inferior lateral corner of coxa (biting edge) lacking pointed tip (Fig. 2G) with pointed tip (Fig. 2G)
Margin of male sternal sulcus S3 with long setae (Fig. 2E) lacking setae (Fig. 2F)
Male sternal sulcus S3/4 reduced to 2 deep lateral notches (Fig. 2E) indiscernible (Fig. 2F)
Table 4.

Comparison of selected morphological characters between two populations (morphotypes) of Louisea nkongsamba from Mount Nlonako, Cameroon.

Characters Morphotype 1 Morphotype 2
Nlonako Engugue1462 Nlonako Eyimba, Ngaltongue, Engugue1382, Nguegue and type specimens
Exorbital tooth relatively large (Fig. 2J, L) relatively small (Fig. 2I, K)
Epibranchial tooth small (Fig. 2J, L) reduced to granule (Fig. 2I, K)
Intermediate tooth between exorbital & epibranchial teeth relatively large (Fig. 2J, L) relatively small (Fig. 2I, K)
Lateral margin posterior to epibranchial tooth lined with small granules (Fig. 2J) smooth (Fig. 2I)
Postfrontal crest poorly defined, completely traversing carapace, reaching anterolateral margins at intermediate tooth (Fig. 2J, L) clearly defined, completely traversing carapace, not reaching anterolateral margins (Fig. 2I, K)
Major cheliped dactylus slim, straight (Fig. 3A) slim, gently arched (Fig. 3B)
Cheliped carpus inner margin teeth distal tooth larger than proximal tooth, both slender, positioned some distance from each other (Fig. 3D) distal tooth larger than proximal tooth, both robust, positioned relatively closer to each other (Fig. 3E)
Medial inferior margin of cheliped merus with small but distinct jagged distal tooth angled outward at 60°, followed by numerous granules and small teeth (Fig. 3I) with large jagged distal tooth angled outward at 90°, followed by numerous granules and small teeth decreasing in size proximally (Fig. 3J)
Mandible inferior lateral corner of coxa (biting edge) lacking pointed tip (Fig. 2P) with pointed tip (Fig. 2O)
Male sternal sulcus S3/4 indiscernible except for 2 deep lateral notches (Fig. 2N) indiscernible, lacking lateral notches (Fig. 2M)
Figure 2. 

Louisea yabassi from Ebo Forest, Cameroon, adult male (CW 20.2 mm) from site no. 1 (A, C, E, G), adult male (CW 21.3 mm) from site no. 2 (B, D, F, H). Louisea nkongsamba from Nlonako, Cameroon, adult male (CW 18.2 mm) from Eyimba (I, K, M, O), subadult male (CW 12.0 mm) from Enguegue (site no. 1) (J, L, M, P). A, B, I, J dorsal view of cephalothorax C, D, K, L frontal view of cephalothorax E, F, M, N ventral view of thoracic sternum G, H, O, P frontal view of left mandible. Scale bars: 8 mm (A, C, E); 9 mm (B, D, F); 1 mm (G, H); 4 mm (I, K); 12 mm (J, M); 3 mm (L); 8 mm (N); 2 mm (O, P).

Figure 3. 

Louisea nkongsamba from Nlonako, Cameroon, subadult male (CW 12.0 mm) from Enguegue (site no. 1) (A, D, I), adult male (CW 18.2 mm) from Eyimba (B, E, J). Louisea yabassi from Ebo Forest, Cameroon, adult male (CW 21.3 mm) from site no. 2 (C, H), adult male (CW 20.2 mm) from site no. 1 (F, G). A, B, F, H frontal view of chela C, D, E, G cheliped carpus I, J cheliped merus. Scale bars: 5 mm (A–J).

The pubertal moult estimates indicate that the largest Louisea species is L. yabassi (CW 24 mm); the smallest species is L. balssi (CW 16.2 mm); while the size ranges of L. edeaensis and L. nkongsamba overlap with each other (~CW 20 mm) in between those of L. balssi and L. yabassi (Table 2). Louisea balssi is a high-altitude species that dwells at 1958 m a.s.l.; L. nkongsamba is a submontane species found between 938 and 1462 m a.s.l.; while both L. edeaensis and L. yabassi are low-altitude crabs, occurring at 90 m a.s.l. and 300 m a.s.l., respectively (see Mvogo Ndongo et al. 2017a, 2017c, 2019, 2021b; Table 2).

Molecular analyses

The present molecular analyses support recognition of three lineages (as population 1, 2, and 3) of L. nkongsamba from six sites on Mount Nlonako (Figs 46). These distinct lineages, however, do not correlate with the two morphotypes recognised herein for L. nkongsamba (Table 4). Population 1 of L. nkongsamba included specimens that were collected from all six localities of Mount Nlonako (Tables 1, 5; Fig. 4); population 2 of L. nkongsamba comprised specimens that were collected from five out of six sites of Mount Nlonako (Tables 1, 5; Fig. 4); and population 3 of L. nkongsamba comprised specimens that were collected from four out of six sites of Mount Nlonako (Tables 1, 5; Fig. 4). Both morphotypes of L. nkongsamba are represented in one or the other population (Table 1).

Table 5.

Number of individuals of Louisea nkongsamba studied per site/population.

Sites Altitude (m a.s.l.) Number of individuals in Population 1 Number of individuals in Population 2 Number of individuals in Population 3
Enguegue no. 2 1382 1 3 1
Ngaltongue no. 1 1176 4 1 0
Ngaltongue no. 2 1256 4 0 1
Enguegue no. 1 1462 1 3 1
Nguegue 1211 3 2 0
Eyimba 938 1 3 1
Total 14 12 4
Figure 4. 

ML tree topology for Louisea species of Cameroon, derived from mtDNA sequences corresponding to three mtDNA loci (partial 12S rRNA, 16S rRNA, and COI genes). BI and ML statistical values (%) on the nodes indicate posterior probabilities and bootstrap support, respectively.

The uncorrected p-distance between Louisea species pairs reveal that each is well isolated from other taxa assigned to this genus (Table 6). Louisea nkongsamba is sister species to L. yabassi with relatively low p-distance (3.97%) (Table 6); both are sister to L. edeaensis. Louisea balssi is isolated from L. edeaensis, with a sequence divergence of 11.04% (12S rRNA), 10.15% (COI), and 7.77% (16S rRNA) (Table 6); from L. yabassi, with a sequence divergence of 12.94% (12S rRNA), 7.32% (COI), and 5.36% (16S rRNA); and from L. nkongsamba, with a sequence divergence of 12.42% (12S rRNA), 7.98% (COI), and 5.04% (16S rRNA) (Table 6). The uncorrected p-distances between the three genetic populations of L. nkongsamba are given in Table 7. Population 1 of L. nkongsamba is sister to population 2, both populations are sister to population 3.

Table 6.

Pairwise uncorrected p-distances of COI, 16S rRNA, and 12S rRNA partial sequences between the species of Louisea.

Louisea species Uncorrected p-distance
COI 16S rRNA 12S rRNA
L. nkongsamba and L. yabassi 3.97% 2.15% 3.77%
L. nkongsamba and L. edeaensis 8.61% 4.33% 4.92%
L. nkongsamba and L. balssi 7.98% 5.04% 12.42%
L. edeaensis and L. yabassi 8.88% 4.35% 4.27%
L. edeaensis and L. balssi 10.15% 7.77% 11.04%
L. yabassi and L. balssi 7.32% 5.36% 12.94%

The phylogenetic analysis indicates that L. balssi from Mount Manengouba is the ancestral species, while L. edeaensis from Lake Ossa is the sister species of the clade that includes L. yabassi and L. nkongsamba (Fig. 4). Divergence time calculations of Louisea species (Fig. 5) showed that the early divergence within the genus occurred during the late Miocene, i.e., L. balssi diverged from other species at about 5.6 myr. Louisea yabassi and L. nkongsamba diverged from L. edeaensis at about 4.1 myr, and L. yabassi separated from L. nkongsamba at about 2.48 myr.

Figure 5. 

BI tree topology for Louisea species of Cameroon, derived from COI mtDNA sequences. Statistical values on the nodes indicate dates in millions of years.

The haplotype network recovered eight haplotypes for L. nkongsamba with maximum four mutation steps between the specimens of this species (Fig. 6) and distinguishes between the four Louisea species (Fig. 6).

Figure 6. 

Maximum parsimony genotype networks for Louisea species of Cameroon, derived from COI mtDNA sequences. Hatch marks stand for mutation steps.

Discussion

Phylogenetic and phylogeographic relationships

The four Louisea species recovered here each has a monophyletic clade (Fig. 4) with strong topological statistical support, and high pairwise uncorrected p-distance values between species pairs (Table 6). This study, therefore, supports the continued recognition of all four Louisea species that are endemic to the southwest Cameroon rainforests. The earliest divergence for Louisea species happened at ~5.6 myr (late Miocene; Fig. 5), which corresponds to the dates for cladogenesis within genera provided by Daniels et al. (2015). In contrast, the latest Louisea divergence between L. nkongsamba and L. yabassi seems to have occurred during the late Pliocene (2.48 myr). Similar divergence times were recovered for another Central African freshwater crab species pair, Sudanonautes aubryi (H. Milne Edwards, 1853) and S. floweri (De Man, 1901) (see Daniels et al. 2015: fig. 3). Even the two morphologically variable Louisea species, L. nkongsamba and L. yabassi, were found in the molecular analyses to have low uncorrected p-distance values (Table 6), but both are recognised as distinct (see Mvogo Ndongo et al. 2019).

Table 7.

Pairwise uncorrected p-distances of COI, 16S rRNA, and 12S rRNA partial sequences between the populations of Louisea nkongsamba.

Louisea nkongsamba Uncorrected p-distance
COI 16S rRNA 12S rRNA
Population 2 and Population 3 0.48% 0.87% 0.95%
Population 2 and Population 1 0.70% 0.20% 0.71%
Population 3 and Population 1 0.52% 0.61% 1.18%

Louisea species are found in different habitats within the rainforest zone: L. balssi in montane forest streams; L. nkongsamba in submontane forest streams; L. edeaensis on the islands of a freshwater lake; and L. yabassi in lowland forest streams. Louisea nkongsamba specimens from cool mountain streams draining the submontane forests of Mt. Nlonako (938–1462 m a.s.l.) are small-bodied with adult males measuring CWs 16–20 mm. Louisea balssi adult males from the cool high-altitude streams (1,958 m a.s.l.) draining into the caldera of Mount Manengouba are also noticeably small-bodied (CWs 13.0–16.2 mm). This agrees with the findings of Daniels et al. (2016) who reported that genetic differentiation tends to be somewhat limited in small-bodied montane species of freshwater crabs. Only a limited genetic variation, however, was found in the lowland forest species, L. edeaensis. In comparison, the moist tropical rainforests surrounding Mount Manengouba receive a high annual rainfall that has maintained a stable forest ecosystem, even during drier periods in the past when rain forests were replaced by savannas in other parts of Central Africa (Brown and Ab’Saber 1979; Diamond and Hamilton 1980; Mayr and O’Hara 1986; Grubb 1992; Zimkus 2009). Consequently, in such high rainfall areas, L. balssi would be sheltered from the harsher effects of rainforest disruption arising from prolonged dry periods in the past, making the Cameroon Highlands a Pleistocene forest refuge for freshwater crab species. Over time, Louisea dispersed from its original location around Mount Manengouba into the surrounding forests of southwest Cameroon, including Mount Nlonako. There L. nkongsamba evolved and continued to disperse into the forested lowlands around Yabassi and Lake Ossa, where L. yabassi and L. edeaensis evolved.

Intraspecific morphological variation

The two L. yabassi populations from localities ~2–3 km apart in the Ebo Forest genetically form a single clade with little lineage differentiation (Fig. 4; populations 1 and 2), and these individuals show relatively low levels of morphological variation (Table 3). Despite this, two L. yabassi morphotypes could be identified (Table 3). Similarly, the six sampled localities around Mount Nlonako, where L. nkongsamba is found, are 4–10 km apart (Tables 2, 5). These individuals of L. nkongsamba fall into three genetically recognisable populations (Fig. 4; populations 1–3), which in turn have two distinct morphotypes (Table 4). Populations 1 and 3 consisted of individuals that all belong to morphotype 1, while population 2 included individuals of both morphotypes (Table 1). The high carapace (CH/FW = 1.3) and narrow front width (CW/FW = 2.9) of both L. yabassi and L. nkongsamba are associated with a semi-terrestrial, air-breathing lifestyle (Cumberlidge 1999). Populations of both species prefer temporary water bodies such as puddles near small permanent streams, as well as damp environments under small stones or in forest floor leaf litter adjacent to streams (Mvogo Ndongo et al. 2017a, 2018, 2019, 2021b). Freshwater crabs have limited dispersal abilities due to the absence of a free-swimming larval phase and their direct development resulting in crab hatchlings; the limited dispersal abilities of the crabs and the restricted movements of the adults in combination with the isolated and fragmentary nature of their wetland habitats might be at least partly responsible for their rich diversity and high endemism (Cumberlidge et al. 2009; Mvogo Ndongo et al. 2021b). The intraspecific morphological and genetic variations observed within L. yabassi and L. nkongsamba are crucial for adaptation by natural selection, not least because low levels of variation are associated with the extirpation of populations and an increased risk of species extinction (Bolnick et al. 2011; Scheiner and Holt 2012; Forsman 2014).

Acknowledgements

We thank the Rufford Small Grant Foundation for funding fieldwork in the South and Southwestern Regions of Cameroon, and the Museum für Naturkunde for funding the first author during a research visit to Germany. We also thank Mr Robert Schreiber and Bernhard Schurian, the Digital and DNA lab managers, respectively, at the Museum für Naturkunde, for their important collaboration during the research visit by the first author to Germany during 2021, and Prof. Alain Didier Missoup for the administrative support of the fifth author at the Zoology Unit, Laboratory of Biology and Physiology of Animal Organisms, Faculty of Science, University of Douala.

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