TY - JOUR
AU - Cumberlidge,, Neil
AB - ABSTRACT Recent taxonomic studies of forest habitats in South Africa have revealed the presence of three new species of freshwater crabs, suggesting that decapod diversity within the indigenous forest biome remain poorly documented. Surveys of the freshwater crabs of north eastern KwaZulu-Natal province produced a number of specimens from Ntendeka Wilderness Area (Ngome forest) and Nkandla and Ngoye forests that proved to belong to two new species following morphological and molecular analysis (mtDNA sequencing of three loci, 12S rRNA, 16S rRNA and COI). Two undescribed species, P. ntendekaensis sp. nov. and P. ngoyensis sp. nov. are described. The divergence time estimation of the new phylogeny for eastern and southern African freshwater crabs is discussed to illuminate biogeographic patterning and to understand factors responsible for cladogenesis. INTRODUCTION The forest biome in South Africa is small, comprising only 0.5% of the total land surface of the country (Mucina & Rutherford, 2009), but it nevertheless supports a high level of species diversity and endemism, particularly among invertebrate taxa. Current levels of species diversity are likely underestimates of true species diversity, given the highly fragmented nature of the forest biome, the lack of recent surveys, especially among habitat-specialist invertebrate groups, coupled with limited taxonomic expertise. The latter observation is corroborated by several recent molecular systematic studies among invertebrate groups that clearly demonstrate the presence of several undescribed species. For example, in the two endemic South African velvet worm genera, Peripatopsis Pocock, 1894, and Opisthopatus Purcell, 1899, eight and five new species respectively have recently been described, while several species still await formal description (McDonald et al., 2012; Daniels et al., 2013, 2016; Ruhberg & Daniels, 2013; Barnes & Daniels, 2019; SRD, unpublished data). In addition, Herbert (2017) recently described a new genus and eight new species of tail-wagger snails (Urocylidae) from South African forests. Three new species of freshwater crabs in the genus PotamonautesMacLeay, 1838 were similarly described from the coastal belt forests of the Eastern Cape and KwaZulu-Natal provinces of South Africa (Peer et al., 2015, 2017; Daniels, 2017). These discoveries underline the fact that South Africa’s under-surveyed forests are likely to harbour several undescribed invertebrate species specifically in poorly sampled or understudied invertebrate groups. This justifies renewed systematic surveys of the country’s forested areas to document biodiversity and to highlight the need to conserve the biome. South Africa has two major forest types: Afrotemperate forests, occurring predominantly in the interior whereas the Indian Ocean coastal belt (IOCB) forests occur predominantly along the eastern coastline (Mucina & Rutherford, 2009). Taxa occupying the Afrotemperate forests generally represent older faunal components, with high numbers of paleoendemic invertebrates whereas those species present in the IOCB forests represent more recent colonization events since this forest habitat is young (Stuckenberg, 1962; Mucina & Rutherford, 2009). While the South African freshwater crab fauna is taxonomically well-studied, the recent discovery of three undescribed forest-endemic species (P. isimangaliso Peer & Gouws, 2015 in Peer, Perissinotto, Gouws & Miranda, 2015, P. mhlopheDaniels, 2017, and P. danielsiPeer, Gouws, Lazo-Wasem, Perissinotto & Miranda, 2017) has led to a renewed interest in the fauna of this biome (Peer et al., 2015, 2017; Daniels et al., 2017). The interior of KwaZulu-Natal is dominated by the Drakensberg Mountains where streams are inhabited by P. clarusGouws, Stewart & Coke, 2000 and P. depressusKrauss, 1843. These two species are part of a deeply-divergent species complex that comprises five cryptic lineages (Phiri & Daniels, 2016). The Mgeni River flows from the Drakensberg Mountains and is inhabited by P. dentatusStewart, Coke & Cook, 1995, while the IOCB forests of KwaZulu-Natal are inhabited by three species, P. lividus Gouws, Stewart & Reavell, 2001, P. danielsi, P. isimangaliso, and the low-lying rivers and streams of the province are dominated by P. sidneyi Rathbun, 1904. Potamonautes sidneyi sensu lato, is broadly distributed from the Eastern Cape to KwaZulu-Natal and Mpumalanga provinces, and is a species complex comprising several distinct lineages (Gouws et al., 2015). Considering that all three of the new freshwater crabs were discovered in forested areas, we initiated a sampling regime of poorly collected forested regions along the coast and interior of KwaZulu-Natal to explore the possible presence of novel lineages. In this respect, a survey of the previously unsampled indigenous forest at Ntendeka Wilderness Area (which includes Ngome forest) in the south-eastern regions of KwaZulu-Natal resulted in the discovery of a bright red freshwater crab that did not conform to any of the described species known to occur in the province. Similarly, a white freshwater crab from Ngoye forest in the IOCB of KwaZulu-Natal contrasted with brown-coloured P. sidneyi found in near sympatry in adjacent streams. Evolutionary relationships between these two colour morphs from Ntendeka Wilderness Area and Ngoye forests, together with other described southern African species of freshwater crabs, were examined to determine if they represent distinct species. It is reasonable to expect that these two colour morphs possibly belong to different species, since Daniels (2017) reported that distinct colour morphs among sympatric freshwater crabs were clearly indicative of species. While the phylogenetics of the southern African freshwater crab fauna is well documented, the factors that are causal to the current biogeographic distribution have not received much attention. Daniels et al. (2002) who produced the first molecular phylogeny for the group, did not undertake a divergence time estimation, while the two subsequent studies (Daniels et al., 2006, 2015) focused on taxonomic questions and biogeographic patterning across the Afrotropical regions. Several undescribed species of freshwater crabs have been described in recent studies (Wood & Daniels, 2016; Peer et al. 2015, 2017; Daniels, 2017) and the time is now opportune to explore the factors causal to cladogenesis among the southern African potamonautid clade, by conducting a divergence-time estimation of the fauna. We examined the evolutionary affinities of the freshwater crab fauna from the forested regions of Ntendeka Wilderness Area (Ngome forest) and Ngoye. It is also hypothesised that the specimens from Ntendeka Wilderness Area and Ngoye forest represent distinct gene pools that are genetically and reproductively isolated. These two undescribed, narrow-endemic, forest-dwelling freshwater crab species are described here. The phylogenetic relationships we generated with the inclusion of the recently described species and the divergence time estimation, are used to explore the cladogenic drivers responsible for the biogeographic distribution among southern African freshwater crabs, with a particular emphasis on the colonization of the forest habitat. MATERIALS AND METHODS Sample collection Crabs were collected from rivers and streams in three localities in the forested regions of KwaZulu-Natal province, South Africa. These included specimens from Ntendeka Wilderness Area (N = 6), Nkandla (N = 4) and, Ngoye forests (N = 4). Eight specimens of a white-crab morph were collected from Ngoye forest (A), and a single specimen of P. sidneyi was present in the adjacent streams of Ngoye forests (B), while all the specimens from Nkandla forest were identified as P. sidneyi (Supplementary material Table S1). DNA extraction, PCR, and sequencing Muscle tissue extracted from walking legs or a single egg, per gravid-female was subjected to DNA extraction using a Nucleospin kit (Macherey-Nagel, Duren, Germany) following the manufacturers protocol. Extracted DNA was stored in a refrigerator at –20oC until required for PCR. Generally, a 1µl DNA in 19µl water dilution was followed prior to use. Three partial mtDNA gene fragments were selected, which included the cytochrome oxidase subunit one (COI), 12S rRNA and 16S rRNA, for the phylogenetic analyses of the newly collected freshwater crabs, These three loci were selected because each has a different mutational rate and has been successfully used for reconstructing evolutionary relationships among freshwater crabs (Daniels et al., 2002, 2006, 2015; Daniels & Klaus, 2018; Gouws et al., 2015; Phiri & Daniels, 2013, 2014, 2016; Wood & Daniels, 2016). Primer pairs are outlined in Daniels et al. (2002, 2006). Standard PCR conditions for amplification and DNA sequencing protocols were followed (Daniels et al., 2006, 2015). Sequences for all 20 of the described South African species of freshwater crabs were downloaded from GenBank for each of the three mitochondrial gene regions and included to validate the phylogenetic placement of the recently-described species (Daniels & Bayliss, 2012; Daniels et al., 2014; Peer et al., 2015, 2017) in relation to the described southern African species (Daniels et al., 2006, 2015). We used four species of LiberonautesBott, 1955 (L. lugbe, Cumberlidge, 1999, L. latidactylus (De Man, 1903), L. nimba,Cumberlidge, 1999, and L. rubigimanusCumberlidge & Sachs, 1989) as outgroups, since the latter genus is sister to Potamonautes based on Daniels et al. (2015). Phylogenetic analyses and divergence-time estimation We used Sequence Navigator (Applied Biosystems, Foster City, CA, USA) to compute a consensus sequence from forward and reverse strands for each of the three gene fragments. No insertions or deletions were evident for the protein-coding COI locus and sequences for this locus were aligned manually. The 12S rRNA and 16S rRNA loci were aligned using Clustal X v 2.1 (Thompson et al., 1997). Since all three partial fragments occur on the mitochondria and are linked, we combined the DNA sequence data for the three fragments into a single data matrix and conducted all analyses on the combined data set. Maximum likelihood (ML) and Bayesian approaches were used to estimate evolutionary relationships. jModelTest (Posada, 2008) was used to obtain the best-fit substitution model for each of the three gene loci (results not shown). These substitution models were used in the partitioned Bayesian analyses. The best-fit maximum likelihood score was chosen using the Akaike Information Criteria (AIC) (Akaike, 1973), as this reduces the number of parameters that contribute little to describing the data by penalizing more complex models (Posada & Buckley, 2004; Nylander et al., 2004). Maximum likelihood analysis was conducted on the concatenated data set in RAxML ver.7.2.7 (Stamatakis, 2006). The robustness of branches of the best ML tree was assessed with 1000 bootstrap replicates using the CAT algorithm for fast bootstrapping whereas the final tree search was conducted under the GTR+Γ model for both partitions as less complicated models are not implemented in RAxML. Bayesian inferences were used to investigate optimal tree space using the program MrBayes ver. 3.2.6 (Ronquist et al., 2012). We ran four Markov chains for each analysis, with each chain starting from a random tree and run for fifty million generations, sampling each chain every 10,000th tree. This process was repeated four times to ensure that trees converged on the same topology. A 50% majority rule consensus tree was generated from the trees retained (after the burn-in trees were discarded using likelihood plots) with posterior probabilities (pP) for each node estimated by the percentage of time the node was recovered. Posterior probabilities values < 0.95 pP were regarded as poorly resolved. Uncorrected “p” distances were calculated for the COI locus among sister species pairs in Paup ver. 4.0b10 (Swofford, 2002) To determine the divergence time estimations for the southern African potamonautid crabs we applied a Yule tree prior and an uncorrelated lognormal relaxed molecular clock after initial test runs (investigating if the standard deviation of the uncorrelated lognormal clock approaches zero), using potamonautid substitution rates (and their standard deviations) as priors that originate from a fossil calibrated phylogeny of the whole family Potamonautidae (Daniels, 2011; Daniels et al., 2015; Daniels & Klaus, 2018): 0.81% per Ma for the rRNA locus (SD = 0.0013; linked clock models), 2.85% per Ma (SD = 0.005) for the COI locus. The maximum clade credibility tree was determined and annotated in TreeAnnotator ver. 2.4.1 (part of the Beast package) after removal of 10% of the trees as burn-in. Morphology Samples were divided into males and females, as freshwater crabs exhibit taxonomic characters for both sexes and males are generally used for species descriptions. The following measurements, in millimetres (mm) were taken with a digital calliper: carapace length (CL), carapace width at widest point (CWW), width of the posterior margin of the carapace (CWP), distance between the postfrontal crest and the anterior margins of the carapace (PFCD), frontal width, measured between the medial margins of the orbits (FW), distance between the exorbital teeth (CWA), carapace height (CH), length, and width of the meri of pereiopods 2 and 5, the length and height of the major cheliped. Samples have been deposited in the South African Museum of Natural History, Iziko Museums of Cape Town (SAM-A). Crabs were photographed with a Canon EOS 6D camera and Canon 100mm f2.8 Macro USM lens (Cannon, Tokyo, Japan). The structure of gonopods 1 and 2 and of the third maxilliped were photographed with the addition of a 31 mm extension tube to the aforementioned setup to increase magnification. RESULTS Phylogenetic placement and estimation of divergence time of the southern African lineages We amplified 316bp, 362bp, and 600bp for the 12S rRNA, 16S rRNA, and COI loci, respectively. The combined mtDNA sequence data comprised a total of 1278bp. The novel sequences for the three loci were deposited in GenBank; 12S rRNA (accession numbers MK 607182-MK 607195), 16S rRNA (accession numbers MK 607196-MK 607209), and COI (accession numbers MK607210-MK607233). Phylogenetic inference methods (BI and ML) retrieved near-identical tree topologies; hence, only the BI topology is shown and discussed (Fig. 1). The BI topology retrieved a monophyletic PotamonautesMacLeay, 1838, comprised of three distinct clades. Two basal East African freshwater crab clades were retrieved (denoted by 1 and 2 on Fig. 1). The first, and most basal East African clade comprised P. obesus A. Milne-Edwards, 1868 sister to P. namuliensisDaniels & Bayliss, 2012, P. choloensisChace, 1953, and P. calcaratusGordon, 1929; while the second East African clade comprised P. raybouldiCumberlidge & Vannini, 2004, sister to P. platynotusCunnington, 1907, P. suprasulcatusHilgendorf, 1898, and P. lirrangensis Rathbun, 1904 and in turn formed a clade sister to P. niloticus H. Milne Edwards, 1837, P. subukiaCumberlidge & Dobson, 2008, P. odhneriColosi, 1924 and P. bellarussusDaniels, Phiri & Bayliss, 2014. The southern African freshwater crabs were monophyletic (3 on Fig. 1). Within the southern African radiation, the small-bodied mountain-living crabs made up two clades, clade one comprising two species from the Drakensberg Mountains (P. clarus and P. depressus) and clade two comprising four species from the Western Cape Fold Mountains (P. parvispinaStewart, 1997 sister to P. parvicorpusDaniels, Stewart & Burmeister, 2001, P. brinckiBott, 1960 and P. tuerkayiWood & Daniels, 2016). The next clade comprised three coastal-forest dwelling species, P. lividus, sister to P. isimangaliso and P. danielsi. In the following clade, P. mhlopheDaniels, 2017 was sister to P. dentatus Stewart, Cook & Coke, 1995 and the latter two species were in turn sister to a clade comprised of tropical highland species (P. gorongosaCumberlidge, Naskrecki & Daniels, 2017, P. mutareensisPhiri & Daniels, 2013 and P. mulanjeensisDaniels & Bayliss, 2012) forming a clade sister to P. flavusjoDaniels, Phiri & Bayliss, 2014. Potamonautes ntendekaensis sp. nov., from Ntendeka Wilderness Area was sister to P. ngoyensis sp. nov., from Ngoye forest (A) and nested equidistant in the latter clade. The latter clade was sister to all the large-bodied riverine species characterised by a conspicuous epibranchial tooth on the anterolateral margin of the carapace (P. bayonianusBrito-Capello, 1864, P. unispinusStewart & Cook, 1998, and P. warreniCalman, 1918), that were sister to species with either a smooth or granulated anterolateral carapace margin (P. granularisDaniels, Stewart & Gibbons, 1998, P. perlatus H. Milne Edwards, 1837, P. barnardiPhiri & Daniels, 2014, and P. barbaraiPhiri & Daniels, 2014) sister to a monophyletic yet phylogenetically structured P. sidneyi. Specimens from Nkandla forest and the one specimen from Ngoye forest (B) all belong to P. sidneyi. Figure 1. View largeDownload slide A Bayesian Inference phylogram and divergence time estimation for the three mtDNA loci (12S rRNA +16S rRNA + COI) for the eastern and southern African freshwater crab Potamonautes. Bootstrap values > 75% are indicated by #, while posterior probability values > 0.95 pP are indicated by an *. A closed circle on a node indicates the presence of a forest-dwelling freshwater crab lineage. Numbers 1 and 2 denote the two East African freshwater clades, while number 3 denotes the southern African clade. Figure 1. View largeDownload slide A Bayesian Inference phylogram and divergence time estimation for the three mtDNA loci (12S rRNA +16S rRNA + COI) for the eastern and southern African freshwater crab Potamonautes. Bootstrap values > 75% are indicated by #, while posterior probability values > 0.95 pP are indicated by an *. A closed circle on a node indicates the presence of a forest-dwelling freshwater crab lineage. Numbers 1 and 2 denote the two East African freshwater clades, while number 3 denotes the southern African clade. The uncorrected sequence distance for the COI locus between P. ngoyensis sp. nov. and P. ntendekaensis sp. nov. was 8.10%, whereas the uncorrected sequence distance between P. ntendekaensis sp. nov. and P. flavusjo was 9.64%, and the uncorrected sequence divergence between P. sidneyi and the near sympatric P. ngoyensis sp. nov. was 14.48%. The maximum uncorrected distance was 6.55% within the monophyletic P. sidneyi clade. Divergence time estimation indicates that cladogenesis within the first basal East African freshwater crab clade occurred 12.00 mya (95% HDP 10.06–14.25 mya), while the divergence in the second East African freshwater clade occurred 10.93 mya (95% HDP 9.86–12.56 mya). Cladogenesis in the monophyletic southern African freshwater crab clade occurred 8.70 mya (95% HDP 7.55–9.92 mya). The divergence between the two Drakensberg Mountain species and the three Western Cape Fold mountain species occurred 6.77 mya (95% HDP 5.52–7.98 mya). The divergence in the second clade of tropical species occurred 6.69 mya (95% HDP 5.81–7.66 mya) within this clade; the divergence of the three forest-dwelling IOCB species (P. lividus, P. isimangaliso and P. danielsi) occurred 3.32 mya (95% HDP 2.57–4.17 mya). In the largely tropical clade, the divergence of P. mhlophe (IOCB species) from its sister species P. dentatus occurred 4.27 mya (95% HDP 3.26–5.26 mya), while the divergence between the two new forest dwelling species P. ntendekaensis sp. nov. from Ntendeka Wilderness Area was sister to P. ngoyensis sp. nov. occurred 2.27 mya (95% HDP 1.73–2.87 mya). All the large bodied freshwater crab species diverged from a common ancestor 3.33 mya (95% HDP 2.71–4.00 mya). SYSTEMATICS Superfamily Potamoidea Ortmann, 1896 Family Potamonautidae Bott, 1970 Subfamily Potamonautinae Bott, 1970 Genus PotamonautesMacLeay, 1838 Type species Thelphusa perlata H. Milne Edwards, 1837, gender masculine, type species by monotypy. Diagnosis Mandibular palp 2 segmented, anterolateral carapace margin lacks intermediate tooth between exorbital, epibranchial teeth; exopod of third maxilliped with long flagellum; terminal article of gonopod 1 0.25-0.33 as long as subterminal segment of gonopod 1; terminal article of gonopod 2 with long flagellum. Remarks Potamonautes has nearly 105 described species (NC, unpublished data). The 15 subgenera designated by Bott (1955) are no longer considered valid taxa based on molecular phylogenetic analyses (Daniels et al., 2002). Distribution The genus is widespread in the Afrotropical region, and is the most species-rich of the genera in Potamonautidae. With the exception of P. niloticus H. Milne Edwards, 1837 and P. berardiAudouin, 1826, which are present in the Nile Valley. The addition of two new species brings the number of South African species of freshwater crabs to 22. Potamonautes ntendekaensis sp. nov. Material examined South Africa. Holotype: SAM-A090201, adult male collected 21 November 2017, Ntendeka Wilderness Area (Ngome forest) (27°49’34.77’’S, 31°25’10.88’’E), KwaZulu-Natal province, South Africa, collected by Theo Busschau. Paratype: SAM-A090202, sub-adult same collection information as holotype. Additional material: SAM-A090203, four gravid adult females, same collection information as holotype. Diagnosis Based on male holotype (adult male CW 37.56 mm other measurements in Supplementary material Table S2). Carapace: highly arched (CH/CL 0.55); postfrontal crest well-defined, complete, lateral ends meeting anterolateral margins; epigastric crests faint, median sulcus between crests short, not forked posteriorly; exorbital, epibranchial teeth reduced to granules; anterolateral margin posterior to epibranchial tooth smooth (Fig. 5A). Third maxilliped: ischium with distinct vertical sulcus (Fig. 4E, F); s3/s4 complete, V-shaped, deep, midpoint almost meeting anterior margin of sterno-pleonal cavity; margins of s4 low, not raised (Fig. 2B). Cheliped: dactylus (moveable finger) slim, highly arched, enclosing oval interspace, with 3 larger teeth interspersed by smaller teeth along length; propodus (fixed finger) with 4 larger teeth interspersed by smaller teeth along length (Fig. 3A–C); carpus inner margin distal tooth large, pointed, proximal tooth reduced to granule (Fig. 3A); medial inferior margin of merus lined by small granules terminating distally at small, low distal meral tooth, lateral inferior margin smooth. G1 terminal article: 1/3rd length of subterminal segment; first third portion straight in line with longitudinal axis of subterminal segment, middle part directed outward at 45°, widened by raised rounded ventral lobe, tip curving sharply upward (Fig. 4A, B). Carapace red in living specimens. Figure 2. View largeDownload slide Potamonautes ntendekaensis sp. nov. showing the colour when collected. Dorsal image demonstrating the red lateral flanks (A); frontal image, demonstrating the red colour (B). This figure is available in colour at Journal of Crustacean Biology online. Figure 2. View largeDownload slide Potamonautes ntendekaensis sp. nov. showing the colour when collected. Dorsal image demonstrating the red lateral flanks (A); frontal image, demonstrating the red colour (B). This figure is available in colour at Journal of Crustacean Biology online. Figure 3. View largeDownload slide Potamonautes ntendekaensis sp. nov. male holotype (CL = 26.50 mm) SAM-A090201: whole specimen, dorsal aspect (A); whole specimen, ventral aspect (B); cephalothorax, frontal aspect (C). Scale line represents 10 mm. Figure 3. View largeDownload slide Potamonautes ntendekaensis sp. nov. male holotype (CL = 26.50 mm) SAM-A090201: whole specimen, dorsal aspect (A); whole specimen, ventral aspect (B); cephalothorax, frontal aspect (C). Scale line represents 10 mm. Figure 4. View largeDownload slide Potamonautes ntendekaensis sp. nov. male holotype (CL = 26.50 mm), SAM-A090201: left gonopod 1, anterior view (A); left gonopod 1 posterior view (B); left gonopod 2 anterior view (C); left gonopod 2 posterior view (D); third maxilliped anterior view (E); third maxilliped posterior view (F). The scale line represents 10 This figure is available in colour at Journal of Crustacean Biology online. Figure 4. View largeDownload slide Potamonautes ntendekaensis sp. nov. male holotype (CL = 26.50 mm), SAM-A090201: left gonopod 1, anterior view (A); left gonopod 1 posterior view (B); left gonopod 2 anterior view (C); left gonopod 2 posterior view (D); third maxilliped anterior view (E); third maxilliped posterior view (F). The scale line represents 10 This figure is available in colour at Journal of Crustacean Biology online. Figure 5. View largeDownload slide Potamonautes ngoyensis sp. nov., colour when collected alive showing the pale-white dorsal aspect. This figure is available in colour at Journal of Crustacean Biology online. Figure 5. View largeDownload slide Potamonautes ngoyensis sp. nov., colour when collected alive showing the pale-white dorsal aspect. This figure is available in colour at Journal of Crustacean Biology online. Description Based on holotype: outline transversely oval, widest anteriorly, narrow posteriorly (CWP/CL 0.67); highly arched (CH/CL 0.55), front broad, measuring one-third CWW (FW/CWW 0.33); urogastric, cardiac, grooves distinct, other grooves faint or missing; postfrontal crest complete, lateral ends meeting epibranchial teeth; epigastric crests faint, median sulcus between crests short, not forked posteriorly; exorbital, epibranchial teeth each reduced to granule; anterolateral margin between exorbital, epibranchial teeth smooth, curving slightly outward, lacking intermediate tooth; anterolateral margin posterior to epibranchial tooth smooth (Fig. 3A–C); branchiostegal wall vertical sulcus faint, meeting longitudinal sulcus, dividing branchiostegal wall into 3 parts, all smooth; suborbital margin faintly granular (Fig. 3B). Third maxilliped: filling entire buccal frame, except for respiratory openings; exopod with long flagellum, ischium with distinct vertical groove (Fig. 4E, F). Epistomial tooth large, triangular, margins lined by large granules (Fig. 3B). Mandible: palp two-segmented; terminal segment simple; tuft of setae at junction between segments. Sternum: s1, s2 fused; s2/s3 deep, completely crossing sternum; s3/s4 complete, V-shaped, deep, midpoint almost meeting anterior margin of sterno-pleonal cavity; margins of s4 low, not raised (Fig. 3B). Cheliped: dactylus (moveable finger) slim, highly arched, enclosing oval interspace, with 3 larger teeth interspersed by smaller teeth along length; propodus (fixed finger) with 4 largest teeth interspersed by smaller teeth along length (Fig. 3C); carpus inner margin distal tooth large, pointed, proximal tooth reduced to granule (Fig. 4A); medial inferior margin of merus lined by series of small granules terminating distally at small, low distal meral tooth, lateral-inferior margin smooth. Pereopods: walking legs slender, pereopod 3 longest, pereopod 5 shortest; dorsal margins of pereopods with fine sharp bristles, dactyli of walking legs each ending in sharp point, with rows of spine-like bristles along segment (Fig. 3A–C). Pleon: outline broadly triangular with straight margins. G1 terminal article: short (1/3 length of subterminal segment), curving away from midline, widest at junction with subterminal segment; first third straight in line with longitudinal axis of subterminal segment, middle part directed outward at 45°, widened by raised rounded ventral lobe, tip pointed, curving sharply upward (Fig. 4A, B); G1 subterminal segment broad at base, tapering to slim junction with terminal article distally where these two parts have same width (Fig. 4A, B), ventral side of segment with setae-fringed flap covering lateral part of the segment; dorsal side of segment smooth, no flap, with broad membrane on the dorsal side of suture marking junction between terminal, subterminal parts. G2: terminal article long, flagellum-like (Fig. 4C, D). Colour The carapace, postfrontal crest margin, pereopods, and chelipeds of live specimens are red that fades to lighter red following preservation in absolute ethanol (Fig. 2A, B). The sternum and pereopods are ventrally red, while the dorsal surfaces of the pereopods are light brown (Fig. 3A–C). Etymology This species is named after the Ntendeka Wilderness area (Ngome forest) to which the species is presently considered to be endemic. The species name is a noun in apposition. Distribution Found under rocks in closed canopy mist belt and coastal scarp forest, away from streams. Remarks Potamonautes ntendekaensis sp. nov. superficially resembles P. flavusjo, P. calcaratus, P. lividus, and P. isimangaliso all of which have a highly vaulted carapace and all of which are semi-terrestrial species that live away from permanent flowing rivers and streams. The dactylus of the right cheliped of adult males of P. calcaratus is arched and conspicuously broad and flat, and there is a single spine-like tooth on the anterolateral margin of the carapace; both characters easily distinguishing the species from P. ntendekaensis sp. nov., P. lividus, P. isimangaliso and P. flavusjo. In these four species the right cheliped dactylus is arched but slim and not flattened, and the anterolateral margin of the carapace is smooth and lacks dentition in all four species. The four semi-terrestrial species are ecologically distinct from each other, despite their common association with ephemeral habitats. Potamonautes flavusjo occurs in seasonal wetland (vlei) areas adjacent to small streams where it burrows into the peat soil of the adjacent grasslands on the Mpumalanga highveld. Potamonautes isimangaliso is endemic to the wetlands around lakes in the False Bay Park region of the iSimangaliso Wetlands Park of coastal KwaZulu-Natal, whereas P. calcaratus occurs around ephemeral pans in the Kruger National Park of South Africa, Mozambique, and neighboring Zimbabwe (Reed & Cumberlidge, 2004). Potamonautes lividus is exclusively associated with the IOCB forests in northeastern KwaZulu-Natal and the Eastern Cape provinces, where it burrows into the soil in forested areas away from freshwater (SRD, unpublished data). Potamonautes ntendekaensis sp. nov. appears to be endemic to the Ntendeka Wilderness forests and it is not known whether it digs burrows, but it is a sister species to P. flavusjo, that is known to burrow. The carapace of live P. lividus is blue with a light blue sheen, and its chelipeds and limbs are orange or red, whereas the carapace of P. flavusjo has distinct yellow markings along the postfrontal crest and the cardiac region while the ventral surface is light yellow to white (Daniels et al., 2014). In contrast, P. isimangaliso has a variable carapace colour when alive, ranging from brown to dark brown, maroon, and almost black, while the tips of the dactyli of its walking legs are either orange or light yellow (Peer et al., 2015). The carapace surface of P. ntendekaensis sp. nov. is bright red (Fig. 2A–C) when alive, fading to dull red when preserved. Potamonautes ntedekaensis sp. nov. is sister to P. ngoyensis sp. nov. and both sister to a clade of highland tropical species. The highland tropical species, P. flavusjo, endemic to the highveld of the Mpumalanga province, South Africa that are sister to a clade comprised of species from the East African mountains, P. gorongosa from Gorongosa National Park in Mozambique, P. mutareensis from the highlands of Zimbabwe, and P. mulanjeensis from Mount Mulanje in Malawi. Adult males of the latter three mountain-living species (and P. ntendekaensis sp. nov.) have a moderately arched right cheliped dactylus, a character shared with most mountain stream freshwater crabs. Neither of the three forest-dwelling, semi-terrestrial freshwater species, P. danielsi, P. lividus nor P. isimangaliso, are closely related to P. ntendekaensis sp. nov. because these species form a more basal branch in the tree topology (Fig. 1). The clear phylogenetic separation of these species suggests that the colonization of forests and the evolution of the semi-terrestrial morphotype in African freshwater crabs happened independently at least three times, and that the convergence in morphology of these species has been driven by habitat similarities, rather than by shared ancestry (Fig. 1). Nomenclatural statement A life science identifier (LSID) number was obtained for the new species: urn:lsid:zoobank.org:pub:1147342C-EF67-4FE2-9210-D0E944D0D0C0. Potamonautes ngoyensis sp. nov. Material examined South Africa. Holotype: SAM-A090204, adult male, collected on 28 January 2018, Ngoye forest (28°50’35.26’’S, 31°42’9.50’’E), KwaZulu-Natal province, coll. Theo Busschau. Paratype. SAM-A090205, sub-adult male, same collection information as holotype. Additional material: SAM-A090206, 1 adult female and five sub adult males, same collection information as holotype. Diagnosis Based on holotype (adult male CWW 32.8 mm; other measurements in Supplementary material Table S3). Carapace: flattened (CH/CL 0.50) (Fig. 5A); postfrontal crest well defined, complete, lateral ends granular, meeting anterolateral margins; epigastric crests faint, medial sulcus between crests short, forked posteriorly; exorbital, epibranchial teeth reduced to granules; anterolateral margin posterior to epibranchial tooth granular (Fig. 6A–C). Third maxilliped: ischium with faint vertical sulcus; s3/s4 complete, V-shaped, deep, midpoint almost meeting anterior margin of sterno-pleonal cavity; margins of s4 low, not raised (Fig. 7E, F). Cheliped: dactylus (moveable finger) slim, highly arched, enclosing oval interspace, 3 larger teeth interspersed by smaller teeth along length; propodus (fixed finger) with 4 larger teeth interspersed by smaller teeth along length; carpus inner margin distal tooth large, pointed, proximal tooth small but distinct, followed by granule; both anterior margins of merus lined by small granules, distal meral tooth small, pointed. G1 terminal segment: 1/3 length of subterminal article; first third straight in line with longitudinal axis of subterminal segment, middle part directed outward at 45°, widened by low, raised, rounded ventral lobe, tip curving gently upward (Fig. 7A, B). Carapace white in living specimens. Figure 6. View largeDownload slide Potamonautes ngoyensis sp. nov. male holotype (CL = 24.50 mm), SAM-A090204: whole specimen, dorsal aspect (A); whole specimen, ventral aspect (B); cephalothorax, frontal aspect (C). Scale line represents 10 mm. This figure is available in colour at Journal of Crustacean Biology online. Figure 6. View largeDownload slide Potamonautes ngoyensis sp. nov. male holotype (CL = 24.50 mm), SAM-A090204: whole specimen, dorsal aspect (A); whole specimen, ventral aspect (B); cephalothorax, frontal aspect (C). Scale line represents 10 mm. This figure is available in colour at Journal of Crustacean Biology online. Figure 7. View largeDownload slide Potamonautes ngoyensis sp. nov. male holotype (CL = 24.50 mm), SAM-A090204: left gonopod 1, anterior view (A); left gonopod 1 posterior view (B); left gonopod 2 anterior view (C); left gonopod 2 posterior view (D); third maxilliped anterior view (E); third maxilliped posterior view (F). The scale line represents 10 mm. Figure 7. View largeDownload slide Potamonautes ngoyensis sp. nov. male holotype (CL = 24.50 mm), SAM-A090204: left gonopod 1, anterior view (A); left gonopod 1 posterior view (B); left gonopod 2 anterior view (C); left gonopod 2 posterior view (D); third maxilliped anterior view (E); third maxilliped posterior view (F). The scale line represents 10 mm. Description Holotype (adult CWW 32.8 mm). Carapace: outline transversely oval, widest anteriorly, narrowest posteriorly (CWP/CL 0.54); flattened (CH/CL 0.50) (Fig. 5A); front broad, one-third CWW (FW/CWW 0.33); urogastric, cardiac grooves distinct, other grooves faint or missing; postfrontal crest complete, lateral ends granular, meeting epibranchial teeth; epigastric crests faint, median sulcus between crests short, forked posteriorly; exorbital, epibranchial teeth each reduced to granule; anterolateral margin between exorbital, epibranchial teeth faintly granulated, curving slightly outward, lacking intermediate tooth; anterolateral margin posterior to epibranchial tooth granulated (Fig. 6A–C); branchiostegal wall vertical sulcus faint, meeting longitudinal sulcus, dividing branchiostegal wall into 3 parts, suborbital, dorsal pterygostomial regions granular, hepatic region smooth; suborbital margin faintly granular (Fig. 6C). Third maxilliped: filling entire buccal frame, except for respiratory openings; exopod with long flagellum, ischium with faint vertical groove (Fig. 7E, F). Epistomial tooth large, triangular, margins lined by large granules (Fig. 6B). Mandible: palp two-segmented; terminal segment simple; tuft of setae at junction between segments. Sternum: s1, s2 fused; s2/s3 deep, completely crossing sternum; s3/s4 complete, V-shaped, deep, midpoint almost meeting anterior margin of sterno-pleonal cavity; margins of s4 low, not raised (Fig. 6B). Cheliped: dactylus (moveable finger) slim, highly arched, enclosing oval interspace, with 3 larger teeth interspersed by smaller teeth along length; propodus (fixed finger) with 4 larger teeth interspersed by smaller teeth along length (Fig. 6A–C); carpus distal tooth large, pointed, proximal tooth small but distinct, followed by granule; both inferior margins of merus lined by small granules, distal meral tooth small, pointed. Pereopods: walking legs slender, pereopod 3 longest, pereopod 5 shortest; dorsal margins of pereopods with fine sharp bristles, dactyli of walking legs ending in sharp point, with rows of spine-like bristles along segment (Fig. 6A–C). Pleon: outline broadly triangular with straight margins. G1 terminal article: short (1/3 length of subterminal segment), curving away from midline, 1/3 length of subterminal segment; first third straight in line with longitudinal axis of subterminal segment, middle part directed outward at 45°, widened by low raised rounded ventral lobe, tip curving gently upward. G1 subterminal segment broad at base, tapering to slim junction with terminal article distally where these two parts have same width, ventral side of segment with heavily setose margins; with setae-fringed flap covering lateral half of segment; dorsal side of segment smooth, no flap, with broad membrane on dorsal side of suture marking junction between terminal, subterminal parts (Fig. 7A, B). G2: terminal article long, flagellum-like, 0.5 times length of subterminal segment (Fig. 7C, D). Colour Carapace of living specimens pearl white (Fig. 5). Etymology This species is named after the forest region of Ngoye in KwaZulu-Natal province in South Africa where the species appears to be endemic. Distribution Found in streams in the Ngoye indigenous coastal forest along the IOCB in close sympatry with P. sidneyi. Remarks The distinguishing characters of P. ngoyensis sp. nov. are the flattened carapace with granular anterolateral margins. This species bears a superficial resemblance to a number of large-bodied southern African species, such as P. sidneyi, P. perlatus, and P. granularis, but was found here to be genetically distinct. Potamonautes ngoyensis sp. nov. is also biogeographically unique because it is a South African forest-dwelling species that may have originated in the tropical forests further north. Our phylogenetic results clearly show that P. ngoyensis sp. nov. is genetically distinct from sympatric P. sidneyi, but the latter is chocolate brown in colour; whereas P. ngoyensis sp. nov. has a distinctive white colour. Colour can also be used to distinguish P. ngoyensis sp. nov. (white carapace) from its sister species P. ntendekaensis sp. nov. (red carapace) in addition to general morphology (a flattened carapace with granular anterolateral margins versus a highly vaulted carapace with smooth anterolateral carapace margins). Potamonautes ngoyensis sp. nov. is superficially similar to P. mhlophe in that both species are pearl-white forest-dwelling species, but the two taxa are phylogenetically distinct (Fig. 1). Nomenclatural statement A life science identifier (LSID) number was obtained for the new species: urn:lsid:zoobank.org:pub:1147342C-EF67-4FE2-9210-D0E944D0D0C0. DISCUSSION Phylogenetics and biogeographic patterning Biotic surveys of the forested regions along the south-eastern coast and interior of the KwaZulu-Natal province, South Africa resulted in the discovery of two new species of freshwater crabs, P. ntendekaensis sp. nov. and P. ngoyensis sp. nov. These results, together with the recent discovery of three new freshwater crab species (Daniels, 2017; Peer et al., 2015, 2017) from forested region in KwaZulu-Natal and the Eastern Cape underline the need for further taxonomic surveys of the forest biome in South Africa. These findings suggest that it may be too early to consider South Africa a well-surveyed country given the strong potential for additional undescribed alpha diversity (especially point endemics) in the forest biome alone. Our dated phylogeny reveals widespread cladogenesis during the late Miocene amongst the deeper nodes of the tree topology (Fig. 1). Our phylogenetic results indicate the presence of two highly divergent, non-sister clades along the East African coast, suggesting two independent colonization events, or alternatively in-situ vicariant speciation in the region. We also clearly observe a low-lying species, or a river dwelling taxon that is basal to a clade of mountain-living species. Our results together with the divergence time estimation of the East African Rift Valley fits well with the uplift of the East African Plateau during the middle Miocene, a factor that was a major cladogenic driver for the freshwater biota of the region (Linder, 2017). Uplifts during the Mio/Plio/Pleistocene created most of the mountain ranges in southern Africa (Clark et al., 2011, Dingle et al., 1983), and were also causal in the isolation and cladogenesis of the freshwater crab fauna. The warm Agulhas current along the southern African coast, together with the rain shadow of the Great Escarpment resulted in the maintenance of a subtropical climate and the persistence of forest patches (Neumann & Bamford, 2015). While Afrotemperate forest patches have persisted here, the development of the proto-Benguela upwelling current during the late Miocene resulted in increased aridification periods (Sieser, 1980; Neumann & Bamford, 2015). These extended episodes of aridification caused allopatric diversification in freshwater crabs and other endemic taxa (Tilbury & Tolley, 2009; Diedericks & Daniels, 2014; Nielsen et al., 2018). Our estimated divergence time supports both a climatic model and a geomorphological shift model to explain cladogenesis in the southern African potamonautid crabs. It is evident from the phylogeny that the forest biome was colonized at least three times by forest-dwelling ancestral freshwater crabs, first during the end of the Miocene, by a lineage that diverged during the middle Pliocene in the three IOCB forest-dwelling sister lineages (P. lividus, P. isimangaliso and P. danielsi), and then for P. mhlophe during the same time period, followed by a recent Plio/Pleistocene divergence between P. ntendekaensis sp. nov. and its sister species P. ngoyensis sp. nov. It is noteworthy that despite the IOCB forest being a young biome (Mucina & Rutherford, 2009) the divergence of the lineages predates the formation of this biome and suggests that the freshwater crabs in the forests were recent colonizers from adjacent areas of either scarp or mistbelt forests in the adjacent interior. Species delineation Convergent evolution among semi-terrestrial species appears to be widespread, and P. ntendekaensis sp. nov. could easily be confused with P. isimangaliso or P. lividus based on morphological similarities. Despite these superficial similarities, however, these three species are clearly phylogenetically distinct (Fig. 1). Similarly, P. ngoyensis sp. nov. is in near sympatry with P. sidneyi in adjacent streams, but the two taxa are both phylogenetically and morphologically distinct. The uncorrected sequence distance between described species of freshwater crabs are high and generally >8.00% for the COI locus (Daniels, 2017; Wood & Daniels, 2016; Daniels & Bayliss, 2012; Daniels et al., 2014). The uncorrected sequence values reported herein compares favourably with those reported during previous studies. Gouws et al. (2015) reported uncorrected sequence divergence values ranging from 2.8% to 14.7% among the clades in the P. sidneyi species complex. As with the recent discovery of P. mhlophe from Mbotyi in the Eastern Cape province, the distinct colour of the two new species was the first indication that these lineages are potentially distinct evolutionary units. Polychromatism in African freshwater crabs remains poorly studied, although this phenomenon is well documented in the Asian freshwater crab fauna (Daniels, 2017). Colour can be highly variable within species, in P. lividus, we observed a light yellow and orange morphotype at the Tugela River mouth; however, these specimens were genetically invariant (SRD, unpublished data), suggesting that colour is not always genetically based but could be the result of environmental factors such as diet and age. We also observed two colour morphs in P. sidneyi, one pearl-white and the other with the regular chocolate-brown morph (SRD, unpublished data). A phylogeographic study of the P. sidneyi species complex is currently being undertaken to delineate species boundaries since multiple lineages have been detected in an earlier study (Gouws et al., 2015). Populations of P. sidneyi in the Eastern Cape and Mpumalanga provinces in the interior of KwaZulu-Natal need to be compared genetically in order to undertake a taxonomic revision of the species. SUPPLEMENTARY MATERIAL Supplementary material is available at Journal of Crustacean Biology online. S1 Table. Localities of material collected. S2 Table. Measurements of holotype and other specimens of Potamonautes ntendekaensis sp. nov. S3 Table. Measurements of holotype and other specimens of Potamonautes ngoyensis sp. nov. ACKNOWLEDGEMENTS A National Research Foundation grant to the first author funded the research and the sample collection. The DNA sequencing facility at the University of Stellenbosch is thanked for performing the sequencing. 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TI - Two new species of freshwater crabs of the genus Potamonautes MacLeay, 1838 (Decapoda: Brachyura: Potamonautidae) from the forests of KwaZulu-Natal, South Africa
JF - The Journal of Crustacean Biology
DO - 10.1093/jcbiol/ruz024
DA - 2019-07-24
UR - https://www.deepdyve.com/lp/oxford-university-press/two-new-species-of-freshwater-crabs-of-the-genus-potamonautes-macleay-dj9hodccgt
SP - 426
VL - 39
IS - 4
DP - DeepDyve
ER -