Journal of Crustacean Biology Advance Access published 10 October 2017

Journal of

The Crustacean Society

Crustacean Biology
Journal of Crustacean Biology 37(6), 723–731, 2017. doi:10.1093/jcbiol/rux087
Version of Record, first published online October 10, 2017, with fixed content and layout in compliance with Art. 8.1.3.2 ICZN.

Sympatric colour morphs or distinct taxa? Examining

species boundaries among two South African
freshwater crabs (Decapoda: Potamonautidae:
Potamonautes MacLeay, 1838), with the
description of a new species
Savel R. Daniels
Department of Botany and Zoology, Private Bag X1, University of Stellenbosch, Matieland, 7602, South Africa

Correspondence: S.R. Daniels; email: srd@sun.ac.za

(Received 13 June 2017; accepted 30 August 2017)

ABSTRACT
A new pearl white freshwater crab was collected in sympatry with Potamonautes sidneyi (Rathbun,
1904) in the Indian Ocean coastal belt forest at Mbotyi on the Wild Coast of the Eastern
Cape Province, South Africa. Specimens of the two sympatric congeneric species were sub-
jected to DNA sequencing of three partial mitochondrial loci (COI, 12S rRNA and 16S
rRNA) together with morphological examination. Phylogenetic analyses of the new species
were undertaken to determine its evolutionary relationship with the other east and southern
African species of freshwater crabs. Results revealed that, Potamonautes mhlophe sp. nov. is sister
to P. dentatus (Stewart & Cook, 1995) and distantly related to the sympatric P. sidneyi. The two
congeneric sympatric species were characterised by the absence of shared COI haplotypes
and marked sequence divergence value for the latter locus. Furthermore, Potamonautes mhlophe
sp. nov. is morphologically distinct from P. sidneyi based on the structure of the first and sec-
ond gonopods. Considering the congruence between the mtDNA and morphology, the new
species is herein described.
Key Words:  12S rRNA, 16S rRNA, cytochrome oxidase 1, molecular evidence, systematics

INTRODUCTION Indigenous forests remain one of the most poorly studied

The taxonomy of the freshwater crabs of southern Africa has
received considerable attention in recent years, resulting in the
description of several new species (e.g., Daniels et al., 2012, 2014;
Phiri & Daniels, 2013, 2014; Peer et  al., 2015, 2017; Wood &
Daniels, 2016; Cumberlidge et al., 2017). The freshwater crab fauna
of South Africa currently stands at 18 described species. As a result
of these recent studies, the freshwater crab fauna of the region
represents one of the best studied in the Afrotropics. Several of
the newly discovered species were found in poorly sampled areas,
such as mountainous regions (i.e., Daniels & Bayliss 2014; Phiri
& Daniels 2013, 2016; Wood & Daniels 2016; Cumberlidge et al.,
2017), while the two most recently described species (Potamonautes
isimangaliso Peer, Perissinotto, Gouws & Miranda, 2015 and P. dan-
ielsi Peer, Gouws, Lazo-Wasem, Perissinotto & Miranda, 2017) were
discovered along the Indian Ocean Coastal belt forest (IOCB) of
the KwaZulu-Natal province, South Africa (Peer et al., 2015, 2017).
biomes in South Africa. The biome is small and highly frag-
mented, and comprises < 0.56% of the land surface of the
country, where it is confined to areas of high precipitation along
the coast and the interior (Mucina & Rutherford, 2006). While
the taxonomic diversity among forest-dwelling invertebrate taxa
is believed to be high, the incomplete taxonomic status of most
forest-dwelling species has hampered accurate estimations of the
alpha diversity and associated conservation efforts. Consequently,
forested regions in the Eastern Cape province of South Africa
has recently become a focal point of a Foundational Biodiversity
Initiate Programme (FBIP) research grant of South Africa, which
aims to document biodiversity and endemism of selected faunal
groups in light of the poor taxonomic status of animals within
this biome. The Eastern Cape province represents a convergence
zone of four major biomes, and harbours high levels of biodiver-
sity (Lubke et al., 1986), with the Maputaland-Pondoland-Albany
region regarded as a biodiversity hotspot (Myers, 2003). The

© Crown copyright 2017.

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 S. R . DANI EL S
indigenous forests of the Eastern Cape Province are mainly com-
prised of Afrotemperate forests in the interior, whereas the Indian
Ocean Coastal Belt (IOCB) forests occurs along the coast (Mucina
& Geldenhuys, 2006). During a recent FBIP-funded sampling trip
to Mbotyi, along the Wild Coast of the Eastern Cape province,
a pearl white freshwater crab was collected in sympatry with
Potamonautes sidneyi (Rathbun, 1904). It remained unclear if the
pearl white species represented a distinct lineage from the sym-
patric rust brown freshwater crab P. sidneyi.
This study was initiated to explore if the pearl white freshwater
crab is genetically distinct from its sympatric congener P. sidneyi by
using mtDNA sequencing, determine the phylogenetic placement
of the pearl white freshwater crab species in relation to the east
and southern African freshwater crabs, and examine if the two
sympatric freshwater crabs are morphologically distinct by com-
paring taxonomically important features. Both the genetic and the
morphological data validate the distinctiveness between the two
sympatric species, hence a new freshwater species is described.
MATERIALS AND METHOD S
Sample collection
Crabs were collected from three localities at Mbotyi, along the
IOCB forest of the Wild Coast of the Eastern Cape province,
South Africa in late September 2016 (Fig. 1). Two groups of speci-
mens could be assigned to P. sidneyi (collected along the outskirts of
Mbotyi (station A; see Fig. 1), the main stream in station B at the
Mqaba forest, included three and seven specimens respectively)
while a pearl white taxon was collected sympatrically in a stream
in the IOCB forest on the outskirts of Mbotyi C and included 14
specimens. A  handheld global positioning system (Garmin-Trek
Summit) was used to obtain coordinates. Specimens were hand
caught from under boulders in streams at all three sample sites
around Mbotyi (stations A, B, and C). Crabs were transported
alive to the field laboratory and killed by freezing at –20  °C for
24  h prior to being preserved in 96% ethanol. Pereopods were
taken from representative samples and the exoskeleton broken to
expose the muscle tissue for presentation in 96% ethanol. Voucher
Figure 1.  Three localities where Potamonautes specimens were collected at Mbotyi, Wild Coast of the Eastern Cape Province, South Africa. The two sample
sites Mbotyi A and B are indicated by ● and represents P. sidneyi, whereas Mbotyi C is indicated by ▲ and represents P. mhlophe sp. nov.
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specimens were deposited at the South African Museum, IZIKO
Museums of Cape Town, South Africa (SAMC-A).
DNA extraction, polymerase chain reaction (PCR) and sequencing
DNA was extracted from the muscle tissue of the ethanol pre-
served pereopod of each specimen (Daniels et  al., 2002) using
a Nucleospin kit (Macherey-Nagel, Duren, Germany), follow-
ing the manufacturers protocol. Extracted DNA was stored
at –20  ºC until required for PCR (Daniels et  al., 2006). Three
mitochondrial markers were sequenced, the cytochrome c
oxidase subunit I  (COI), 12S rRNA, and 16S rRNA using
the primer pairs outlined in Daniels et  al. (2002). These three
markers have been extensively used in phylogenetic studies of
Afrotropical freshwater crabs (Daniels et  al., 2002, 2006, 2015;
Phiri & Daniels, 2014, 2016; Wood & Daniels, 2016). All sam-
ples were sequenced for the COI locus, whereas a single repre-
sentative of the pearl white morphotype was sequenced for the
12S rRNA and the 16S rRNA. The phylogenetic position of
the pearl white morphotype was determined by including data
from the present study with the all the recently described east-
ern and southern African freshwater crab phylogenies (Daniels
et al., 2012, 2014; Phiri & Daniels, 2013; Peer et al., 2015, 2017;
Wood & Daniels, 2016).
A 25  μl reaction solution for each sample was utilised for the
PCR amplifications: Millipore water (14.9  μl), 25  mM MgCl2
(3.5 μl), 10×Mg2+ -free buffer (2.5 μl), 10 mM deoxyribonucleotide
triphosphate solution (0.5 μl), 10 mM forward and reverse genetic
marker primers (0.5 μl each), and 0.1 U Taq polymerase (0.1 μl),
2.5  μl of the 1:19 template DNA dilution. For the target marker
COI, the PCR conditions were 94  ºC (4  min.), (94  °C (30  sec.),
42  ºC (40  sec.), 72  ºC (45  sec.)) for 36 cycles, and a final exten-
sion at 72 ºC (10 min.). The annealing temperature for 12S rRNA
and 16S rRNA was 48 ºC and 50 ºC, respectively. PCR products
were electrophoresed (3 hours in a 1.5% agarose gel containing
ethidium bromide) and the PCR products were then gel purified
with the BioFlux purification kit (Bioer Technology, Alameda, CA,
USA). Sequencing was performed at the Central Analytical Facility
of the Stellenbosch University, Stellenbosch, South Africa.
 A   N E W SP E C I E S OF F R ES HWAT ER CR AB FRO M S O UT H AFR I CA
Network analyses
DNA sequences were aligned and edited using Clustal X
(Thompson et  al., 1997). To check for stop codons in COI
sequences were translated to amino acids using the online pro-
gramme EMBOSS-Transeq (http://www.ebi.ac.uk/emboss/
transeq/); no stop codons were detected. Haplotype networks
were constructed for the three populations of freshwater crabs
collected around Mbotyi (localities A, B, and C) using the rap-
idly evolving COI locus in TCS version 1.3 using a 95% connec-
tion limit (Clement et  al., 2000). Uncorrected pairwise sequence
divergence (“p” distances) for the COI locus were calculated in
PAUP*4 v.beta 10 (Swofford, 2002).
Phylogenetic analysis
At the population level, we conducted phylogenetic analyses exclu-
sively on the COI data for the three Mbotyi sample sites (A, B, and
C) (Fig.  1) using two large bodied South African freshwater crabs
(P. warreni Calman, 1918 and P. unispinus Stewart & Cook, 1998), as
outgroups. This initial phase was followed by a phylogenetic analy-
sis of examples of eastern and southern African freshwater crabs to
determine the phylogenetic placement of the pearl white species.
For the latter portion of the study four species of Liberonautes Bott,
1955 (L.  lugbe Cumberlidge, 1999, L.  latidactylus (De Man, 1903),
L.  nimba, Cumberlidge, 1999, and L.  rubigimanus Cumberlidge &
Sachs, 1989) were used as outgroups since the latter genus is sister
to Potamonautes MacLeay, 1838 based on Daniels et al. (2015).
Maximum parsimony (MP), maximum likelihood (ML), and
Bayesian inference (BI) were used to estimate evolutionary relation-
ships. MP analysis was conducted in PAUP*4 v.beta 10 (Swofford,
2002). For MP analysis trees were generated using the heuristic
search option with TBR branch swapping and 100 random addi-
tions of taxa. Phylogenetic confidence in the nodes recovered from
MP was estimated by bootstrapping, analysing 1000 pseudoreplicates
of data sets (Felsenstein, 1985). The programme jMODELTEST 2
(Darriba et al., 2012) was used to obtain the best-fit substitution mod-
els for the three mtDNA loci using the Akaike information criterion
(AIC) (Akaike, 1973). ML analyses were performed using RAxML
v.7.3.1 (Stamatakis, 2006; Stamatakis et  al., 2008) as implemented
on the CIPRES portal (http://www.phylo.org/subsections/portal/)
(Miller et al., 2010). Rapid bootstrap analysis model and the GTR +
I + G model were selected for the likelihood analysis. A set of 1000
bootstrap replicates was run. Nodes with bootstrap values of < 75%
were considered to be poorly resolved.
Bayesian inferences were used to optimal tree space using
MRBAYES investigate 3.0b4 (Ronquist & Huelsenbeck, 2003).
Ten Markov chain Monte Carlo (MCMC) iterations were run for
10 × 106 generations, with each chain starting from a random tree
and sampling from the chain every 5000th generation. The pro-
gramme AWTY (Nylander et  al., 2008) was used to summarized
the post burnin. Posterior probability (pP) < 0.95 were considered
to be poorly resolved. FigTree v.1.3.1 (Drummond et al., 2009) was
used to view and edit consensus trees.
Morphology
Samples were divided into males and females and the following
measurements were taken with digital vernier callipers: carapace
length (CL); carapace maximum width measured as the width
at the 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); the frontal width
measured between the medial margins of the orbits (FW); distance
between the exorbital teeth (CWA) and the carapace height (CH);
pereiopod measurements were also taken as length and width of
the merus of pereiopods 2 and 5, and the length and height of
the major cheliped. All measurements are given in millimetres
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(mm). Both live and newly preserved crabs, male gonopods 1 and
2, mandibular palp and the maxilliped were photographed with
a Canon 70D (city and country of manufacturer) digital camera.
RESULTS
Phylogenetic analyses
The amplified COI fragment for the 17 Mbotyi specimens was 447
base pairs in length. These new sequences have been deposited in
GenBank (accession numbers for Potamonautes sidneyi, MF693160 -
MF693166; the white freshwater crab Mbotyi C, MF693167 -
MF693176). All three analytical methods (MP/ML/BI) retrieved
identical tree topologies hence only the BI topology is shown and
discussed. For MP, 68 characters were parsimony informative, a
single tree was recovered with a tree length of 77 steps, a con-
sistency index (CI) of 0.96, and a retention index (RI) of 0.99.
The phylogenetic analyses of the COI data for the three Mbotyi
sample sites (A, B, and C) retrieved two statistically well-supported
clades (> 75% / 0.95 pP). Clade 1 comprised P. sidneyi specimens
from sample sites (Mbotyi A  and B), whereas clade 2 was exclu-
sively comprised of the pearl white freshwater crab specimens
(Mbotyi C), Potamonautes mhlophe sp. nov. (Fig. 2). The haplotype
networks (results not shown) retrieved a single haploclade for the
two P.  sidneyi populations and the single pearl white freshwater
crab population respectively. No haplotypes were shared between
clades, corroborating the genetic isolation of the two sympat-
ric gene pools. The uncorrected ‘p’ sequence divergence for the
COI locus between P. mhlophe sp. nov. and P. sidneyi was 13.42%.
The high uncorrected sequence divergence value corroborates
the genetic distinctiveness of the two sympatric freshwater crab
lineages.
For the phylogenetic analyses of eastern and southern African
taxa, the amplified COI, 12S rRNA, and 16S rRNA fragments
were 447 bp, 338 bp, and 517 bp, respectively. The combined
mtDNA data set comprised 1292 bp. Newly generated sequences
were deposited in GenBank (12S rRNA accession number
MF693158; 16S rRNA accession number MF693159). The DNA
substitution models are provided in Table S1 (Supplementary
material). All three analytical methods produced identical tree
topologies, hence only the BI tree is shown and discussed. A total
of 417 characters were found to be parsimony informative, the RI
was 0.54, the CI 0.34, with a total of four trees retrieved and a tree
length of 2,016 steps. The Bayesian analyses retrieved a mono-
phyletic Potamonautes as well as a monophyletic southern African
clade, with most of the eastern African species forming a clade
containing the early branching in the genus (> 75% / pP > 0.95)
(Fig. 3). Within the southern African freshwater crab clade spe-
cies, the montane species were basal in the tree topology, with the
two Drakensberg species (P. clarus Gouws, Stewart & Coke, 2000
and P. depressus Krauss, 1843) forming a clade, whereas the four
Cape Fold Mountains species formed a clade (P. parvispina Stewart,
1997 from the Cederberg Mountains basal to the three Cape Fold
Mountain species P. brincki Bott, 1960, P. parvicorpus Daniels, Stewart
& Burmeister, 2001, and P. tuerkayi Wood & Daniels, 2016). The
following clade was comprised of the KwaZulu-Natal swamp and
coastal forest species, P. lividus (Gouws, Stewart & Reavell, 2001)
sister to the two recently described species (P. danielsi and P. isiman-
galiso) (> 75% / pP > 0.95). Potamonautes mhlophe sp. nov. (from
Mbotyi C, Fig. 2) was retrieved sister to P. dentatus (see Stewart,
Coke & Cook, 1995) (>75% / pP > 0.95). The latter clade was
in turn sister to species from the Highveld of Mpumalanga (P.
flavusjo Daniels, Phiri & Bayliss, 2014) and the mountain living
species in Malawi (P. mulanjeensis Daniels & Bayliss, 2012) and the
recently described P. gorongosa Cumberlidge, Naskrecki & Daniels,
2017 from Mozambique, and P. mutareensis Phiri & Daniels,
2013 from the Zimbabwean Highlands (>75% / pP > 0.95).
 S. R . DANI EL S

Figure  2.  Bayesian phylogram based on the COI locus for the Potamonautes specimens collected from Mbotyi, Eastern Cape Province, South Africa.
Statistical values above the nodes represent values for bootstrapping for maximum parsimony (MP) and maximum likelihood (ML) and the posterior prob-
ablity (pP) values for the Bayesian analyses. Values above each node represent the bootstrapping values while values below each node represent the pP values.
Only bootstrap values > 75% and pP values > 0.95 are shown.

All the large-bodied, river-dwelling South African freshwater
crabs formed a distinct clade sister to the mountain-living clade,
with all the species characterised by dentition on the anterolateral
margins of the carapace forming a clade (P. warreni Calman, 1918;
P. unispinus Stewart & Cook, 1998, and P. bayonianus Brito-Capello,
1864) sister to a clade (>75% / pP > 0.95) of species that lack
dentition on the anterolateral margin of the carapace (P. sidneyi sis-
ter to P. perlatus H. Milne-Edwards, 1837; and P. barbarai Phiri &
Daniels, 2014; and P. barnardi Phiri & Daniels, 2014; and P. granula-
ris Daniels, Stewart & Gibbons, 1998).
Male gonopod 2.  In P. sidneyi, gonopod 2 exhibits a curved terminal
segment (Fig. 4E), which is notably distinct from the straight terminal
segment characteristic of P. mhlophe sp. nov. (Fig. 4F). The overall
morphology, however, is highly conserved in these two species.
Maxilliped and mandibular palp. The structure of the third
maxilliped is the same for Potamonautes sidneyi and P.  mhlophe sp.
nov. (Fig. 4G, H). The mandibular palp is two-segmented and the
terminal segment is single, undivided with setae at the junction
between segments (Fig. 4I–L).

Morphology
Male gonopod 1.  The male gonopod 1 of P. sidneyi is characterised by
a smooth terminal segment that curves outwards at the midpoint in
posterior view. The widest point is basally, with the gonopod ending
in a slightly curved, pointed tip (Fig. 4A, B). The morphology of the
gonopod 1 is conserved, although in P. mhlophe sp. nov. (Fig. 4C,
D) the terminal segment appears to be shorter and slightly more
curved at the tip than in P. sidneyi.
DISCUSSION
Both the genetic and morphological results were congruent and
indicate that the two sympatric freshwater crabs collected at
Mbotyi, represent two distinctive gene pools. The most obvious
difference between the two species is the striking colour difference.
Colour can be highly variable as a diagnostic tool in freshwater
crabs. P. mhlophe sp. nov. can be further characterised based on
the COI data and the combined mtDNA sequence data, high
sequence divergence values for COI, and the traditional alpha

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 A   N E W SP E C I E S OF F R ES HWAT ER CR AB FRO M S O UT H AFR I CA

Figure 3.  Bayesian phylogram for the combined mtDNA dataset (COI + 12S rRNA and 16S rRNA) for the eastern and southern African Potamonautes.
Statistical values above the nodes represent values for bootstrapping for maximum parsimony (MP) and maximum likelihood (ML) and the posterior prob-
ablity (pP) values for the Bayesian analyses. Values above each node represent the bootstrapping values while values below each node represent the pP values.
Only bootstrap values > 75% and pP values > 0.95 are shown. An asterisk (*) indicates nodes that were not statistically supported.

taxonomic characters used in the description of freshwater crab
species. Collectively, these characters provide strong evidence that
the pearl white crab taxon represents an undescribed species.
The phylogenetic placement of P. mhlophe sp. nov. relative to
its sympatric congener, P. sidneyi, further supports the genetic dis-
tinctiveness of the two species. The new species was retrieved as
sister to P. dentatus from the Mgeni drainage in the KwaZulu-Natal
highlands, whereas P. sidneyi was sister to the other large bodied
freshwater crabs. The sister relationship between P. dentatus from
KwaZulu-Natal and P. mhlophe sp. nov. from Mbotyi C suggests
a previously widely distributed subtropical ancestor that has sub-
sequently become extinct, supporting allopatric speciation for
the two current sympatric species. Potamonautes sidneyi is, however,
a species complex in need of taxonomic revision (Gouws et al.,
2015). Gouws et al. (2015) more recently demonstrated the pres-
ence of seven divergent independent networks within the P. sid-
neyi species complex, and described the southern clade (clade E in
Gouws et al., 2015) as a distinct species, P. danielsi. In a recent rede-
scription of P. sidneyi sensu stricto, Peer et al. (2017) restricted the
distribution of the species to north of Durban along the coast to
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Lake Sibaya (representing clades A and B in Gouws et al., 2015).
In the phylogenetic analyses, P. danielsi is sister to P. isimangaliso and
P. lividus, and is not closely allied with P. mhlophe sp. nov. In the
phylogenetic analyses (Fig. 3), the former three species formed a
clade that are geographically restricted the Indian Ocean Coastal
Belt forests of KwaZulu-Natal, whereas P. mhlophe sp. nov. is
restricted to the southern IOCB forests of the Eastern Cape at
Mbotyi. Peer et al. (2017) noted that P. danielsi occurs from the
Mhlanga (Durban North) to the Mtamvuna rivers at the northern
borders of Pondoland based on the genetic work by Gouws et al.
(2015). Considering that P. sidneyi is widely distributed in south-
ern Africa, ranging from the Eastern Cape into KwaZulu-Natal,
Free State, Northern Cape, Mpumalanga, and the Limpopo prov-
inces in South Africa into neighbouring Swaziland and southern
Mozambique, extensive geographic sampling will be required to
delineate species boundaries within the P. sidneyi species complex
(Cumberlidge & Daniels, 2007; Cumberlidge et al., 2017; Gouws
et al., 2017). G. Gouws (pers. comm.) is undertaking a phylogeo-
graphic study of the P. sidneyi species complex to further elucidate
such boundaries. The specimens of P. sidneyi used by Daniels et
 S. R . DANI EL S
al. (2002) (also used herein) is from Empangeni, KwaZulu-Natal
province and is within the distribution range of P. sidneyi sensu
stricto (Peer et al., 2017).
Polychromatism in the Afrotropical Potamonautidae is not well
documented and does not appear to be particularly common. In
contrast, this phenomenon is well known in Potamidae. For exam-
ple, in the Japanese freshwater crab, Geothephusa dehaani (White,
1847), two colour morphs, dark and blue, have been observed,
and these populations exhibit substantial genetic differences based
on five allozyme loci (Aotsuka et  al., 1995). Similarly, Hsi-Te et  al.
(2007) reported that the colour variation in both Geothelphusa olea
(Shy, Ng & Yu, 1994) and Geothelphusa pingtung in southwestern
Taiwan does not appear to be supported by genetic differences for
the COI locus and most likely represent environmental conditions,
suggesting that colour does not have taxonomic value. These stud-
ies suggest that colour differences need to be interpreted with cau-
tion. The inverse is also true, however, and invariance in colour
may frequently mask distinct cryptic lineages. For example, in both
the P. perlatus and P. sidenyi species complexes, all the clades were rel-
atively constant in colour, being a typical rust or chocolate brown
(Phiri & Daniels, 2014; Gouws et al., 2015). Colour is nevertheless
an excellent indicator of species where distinct freshwater colour
morphs are presents and in close geographic proximity with distinct
habitat preferences. For example, Daniels et al. (2014) observed that
in the burrowing freshwater crab, P. flavusjo, which is endemic to the
Highveld of Mpumalanga in South Africa, the margins of the post
frontal crest, chelipeds, and pereiopods had a bright sulphur yellow
colouration, whereas the river-dwelling P. sidneyi found in the same
locality was typically rust brown. Similarly, in the P. clarus-P. depressus
species complex, Phiri & Daniels (2016) observed several distinct
colour morphs among the six observed clades.
The uncorrected sequence divergence for COI between P. mhlophe
sp. nov. and P.  sidneyi is 13.42%. This value is very high when
compared to other taxonomic studies. Gouws et al. (2015) observed
uncorrected sequence divergence values for the COI locus rang-
ing from 2.8% to 14.7% while examining species boundaries in
the P.  sidneyi species complex. Peer et  al. (2015) similarly reported
uncorrected sequence divergence values of 7.9% between P.  lividus
and P.  isimangaliso. Phiri & Daniels (2014) demonstrated the pres-
ence of three cryptic lineages within P.  perlatus (H. Milne Edwards,
1837). These three clades showed very distinct drainage phylopatry,
indicating that freshwater river crabs exhibit limited dispersal capa-
bilities. These three clades could also be separated by marked uncor-
rected COI sequence divergence values. Clade 1A (Gamtoos and
Gouritz drainage systems) and clade 1B (Berg and Breede drainage
systems) showed a mean of 10.7% difference, while clades 1A and
2 (Berg, Eerste, and Olifants rivers and Cape Peninsula) showed a
mean of 10.7%, whereas it was 4.8% between clade 1B and clade
2. Similarly, three allopatric clades were more recently identified in
the Seychelles freshwater crab, Seychellum alluaudi (A. Milne-Edwards
& Bouvier, 1893), and uncorrected COI sequence divergence values,
clades one (from Silhouette Island) were 8.20% different from clades
two and three, whereas clade two (from Praslin Island sister to La
Digue Island) was 4.01% divergent from clade three (Mahe Island
sister to Fregate Island) (Daniels, 2011). Two of these clades were
described as new species of Seychellum Ng, Števčić, and Pretzmann,
1995 (Cumberlidge & Daniels, 2014). It is clear from the above stud-
ies that sequence divergence values for the COI between pairs of
species of freshwater crabs are highly variable. The high uncorrected
COI sequence divergence value observed between the sympatric
potamonautid crabs at Mbotyi, however, falls at the higher end of
these values indicating their evolutionary distinctiveness.
The two freshwater crab species collected at Mbotyi (P. sidneyi and
P. mhlophe sp. nov.) are morphologically distinct. The P. sidneyi speci-
mens conform to the typical P. sidneyi and are characterised by the
chocolate brown to light brown form. Similarly, male gonopods 1
and 2 of the Mbotyi specimens at sample sites (A and B) conform
to those observed in the redescription of P. sidneyi (Peer et al., 2017).
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The absence of major carapacial differences between the two sym-
patric species suggest that cladogenesis in freshwater crabs appear to
frequently occur without altering carapace features, suggesting that
the lack of morphological features almost certainly impact alpha-
taxonomy designations negatively, resulting in an underestimation
of species diversity among species of freshwater crabs. Similar
observations have been made by Phiri & Daniels (2014) while dif-
ferentiating the three species in Potamonautes perlatus sensu lato. The
present results suggest that even in countries such as South Africa
where the alpha taxonomy of freshwater crabs has received signifi-
cant attention, poorly-sampled biomes such as forests and moun-
tainous areas will continue to yield novel species. Similar patterns
are likely to exist throughout the Afrotropical region and should
form the basis of strategic systematic sampling of these regions.
SYSTEMATICS
Superfamily Potamoidae Ortmann, 1896
Family Potamonautidae Bott, 1970
Subfamily Potamonautinae Bott, 1970
Genus Potamonautes MacLeay, 1838
Type species:  Thelphusa perlata H.  Milne Edwards, 1837, gender
masculine, type species by monotypy.
Diagnosis:  Mandibular palp 2 segmented, anterolateral margin
lacks intermediate tooth between exorbital, epibranchial teeth;
exopod of third maxilliped with long flagellum; terminal article of
gonopod 1, 0.25 to 0.33 as long as subterminal segment of gonopod
1; terminal article of gonopod 2 with long flagellum.
Remarks: Potamonautes has nearly 103 described species (N.
Cumberlidge pers. com). The 15 subgenera designated by Bott
(1955) are no longer considered valid taxonomic units 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 two species of freshwater crabs (P.  niloticus
(H. Milne Edwards, 1837) and P. berardi (Audouin, 1826), which is
present in the Nile valley, most species occur south of the Sahara.
The addition of P. mphlophe sp. nov. brings the number of South
African freshwater crab species to 19.
Potamonautes mhlophe sp. nov.
(Figs. 4 C, D, F, H, K, L; 5 A; 6 A–C)
Material examined:  South Africa. Holotype. SAMC-A089029, male
holotype (CL  =  21.05  mm), 28 September 2016, 31°28.359”S,
29°42.716”E, outskirts of Mbotyi in Indian Ocean Coastal
Belt Forest, Wild Coast, Eastern Cape province, South Africa,
collected by S.R Daniels, T.  Busschau, and J.  Kushata. Paratype
SAMC-A089030; male – data as for the holotype. Additional
material examined. SAMC-A089031-44, 7 females and 5 males,
same date, locality and collectors as for holotype.
Diagnosis:  Measurements for P.  mphlophe sp. nov. in Table  1.
Carapace flat, narrow, posteriorly. Postfrontal crest complete
including behind orbits. Epibranchial teeth absent. In life dorsal,
ventral carapace, pereopods pearl white with light mauve sheen.
Dactylus of major cheliped arched with row of small teeth. Pearl
white colouration of living P. mphlope sp. nov. (Fig. 5A) specimen in
sympatry with rust to chocolate brown P. sidneyi (Fig. 5B).
Description:  Male (holotype). Carapace, limbs pearl white when
alive, fades to white when preserved in ethanol (Fig.  6A–C).
Cephalothorax ovoid, distinctly arched, maximum height, width
 A   N E W SP E C I E S OF F R ES HWAT ER CR AB FRO M S O UT H AFR I CA
at anterior third (CH/CL  =  0.42, CWW/CL  =  1.30). Carapace
smooth, urogastric, cardiac groove moderately deep. Exorbital teeth
blunt, epibranchial teeth absent. Anterolateral margin between
exorbital teeth, postfrontal crest finely granular, lacking teeth.
Postfrontal crest poorly developed posterior to eye orbits. Sternites
1, 2 fused, no suture present. Suture between sternites 2, 3 complete,
deep suture between sternites 3, 4 complete, shallow lateral part
sloping gently towards pleon. Second sulcus prominent, running
completely across sternum, third sulcus projecting downwards
medially to abdominopelvic region. Sulci, episternal sulci thereafter
well defined but shallow. Third maxillipeds filling entire buccal
frame except for small, oval respiratory opening. Ischium scabrous,
with wide groove running vertically. Flagellum on exopod long.
Figure  4.  Potamonautes sidneyi, male: left gonopod 1, posterior view (A);
left gonopod 1, anterior view (B). P.  mhlophe sp. nov., male holotype
(SAMC-A089029), Mbyoti, Wild Coast, Eastern Cape Province, South
Africa: left gonopod 1 posterior view (C); left gonopod 1, anterior view (D).
P. sidneyi, male: left gonopod 2, posterior view (E). P. mhlophe sp. nov., male
holotype (SAMC-A089029): left gonopod 2, posterior view (F). P. sidneyi: left
maxilliped (G). P. mhlophe sp. nov.: left maxilliped (H). P. sidneyi: left man-
dibular palp anterior view (I). P. sidneyi: left mandibular palp, posterior view
(J). P. mhlophe sp. nov.: left mandibular palp anterior view (K). P. mhlophe sp.
nov.: left mandibular palp, posterior view (L). Scale bar = 1.0 cm for A–H,
0.5 mm for I–L.
Table 1.  Potamonautes mhlophe sp. nov. Measurements (in mm) of the holotype and ranges for males and females examined.
Variable
carapace length
carapace width at widest point
carapace posterior margin
frontal width
distance between postfrontal crest and anterior margin
carapace height
major cheliped propodus length
pereiopod 2, merus length
pereiopod 2, merus width
pereiopod 5, merus length
pereiopod 5, merus width
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Mandibular palp with 2 segments, terminal segment characterised
by small ledge at junction of segments, with setae along the margin.
Subterminal segment enlarged distally then compressed at joint with
terminal segment. Chelipeds markedly unequal, enlarged arched
right dactylus. Chelipeds heterochelous, right cheliped larger than
left cheliped. Pereopods slender. Carpus on either side containing
prominent tooth followed by small tooth. Meri strongly granular
at margins. Gonopod 1, terminal segment short, approximately
0.25 length of subterminal segment (Fig. 4D–F). Terminal segment
curves away from mid-line when viewed posteriorly, widest at basal
point, ending in pointed tip. Subterminal segment tapering distally,
inner margin slightly irregular. Basal margins of subterminal
segment 1 with setae. Gonopod 2 hollow, approximately 0.4 length
of subterminal segment, widest at base, tapering sharply inward
at 0.3 times length, forming narrow upright process supporting
terminal segment, terminal segment markedly curved (Fig. 4D–F).
Etymology:  Named using the iSiXhosa word mhlophe for ‘white,’ with
reference to the pearl-white colour of the species. The iSiXhosa
people are the traditional stewards of the Wild Coast in the Eastern
Cape province of South Africa. The name is used as a noun in
opposition.
Distribution:  Potamonautes mhlophe sp. nov. is so far restricted to a
narrow Indian Ocean coastal-belt forest where it occurs under
boulders in the streambed on the outskirts of Mbotyi, Wild
Coast of the Eastern Cape province, where it is sympatric with
P. sidneyi. Further sampling of the forest patches along the coastal
trip southwest of Mbotyi should be examined for the occurrence
of the new species. Potamonautes mhlophe sp. nov. is of immediate
conservation because of its apparent narrow distribution range,
land it appears to be endangered using the IUCN criteria.
Remarks:  Despite the superficial similarities between P. sidneyi and
P. mhlophe sp. nov. the two species are genetically highly divergent.
The most obvious difference is the colour difference between the
two sympatric species. The two species are also very different
in the structure of their male gonopods 1 and 2 (Fig.  4A–F).
Potamonautes mhlophe sp. nov. is diagnosed primarily on the basis
of genetic evidence, a distinct colour difference, and differences
in the gonopod morphology. The new species is also superficially
similar to other montane species of South African freshwater
crabs but it can be distinguished on the basis of species-specific
characters. P. mhlophe sp. nov. is distinct from its sister P. dentatus
as it lacks dentition on the anterolateral margins of the carapace,
and a notch on the post frontal crest posterior to the exorbital
tooth. The two species are also ecologically distinct, with P. dentatus
living in medium to large rivers ranging from 8 to 40 m wide,
and in water depths of 10 to 50cm at altitudes of 355 to 1115
m above sea level (Stewart et  al., 1995), whereas P.  mhlophe sp.
nov. lives in shallow coastal streams. In addition, P. dentatus is dark
Holotype Males Females
21.05 14.09–23.56 13.07–24.50
29.11 19.34–33.68 19.20–34.45
10.62 6.05–10.78 6.08–12.20
20.21 12.20–20.22 13.45–22.06
3.01 2.20–3.09 2.01–2.90
9.05 5.52–11.66 4.15–10.26
27.10 12.33–32.68 7.56–22.89
10.25 6.05–12.81 5.67–12.82
3.04 2.42–3.11 2.11–3.56
10.71 10.12–13.45 6.75–12.13
2.35 2.01–3.29 2.42–3.06
729
 S. R . DANI EL S
Figure 5.  Dorsal images of the two live freshwater crab species to dem-
onstrate the marked colour differences. Potamonautes mhlophe sp. nov. male
paratype (SAMC-A089030), has a characteristic pearl white colour (A); P.
sidneyi male is rust brown (B). Scale bar = 1.5 cm. This figure is available in
colour at Journal of Crustacean Biology online.
Figure 6.  Potamonautes mhlophe sp. nov. male holotype (SAMC-A089029),
Mbotyi, Wild Coast, Eastern Cape Province, South Africa (CL = 21.05
mm): whole animal, dorsal view (A); whole animal ventral aspect (B); cepha-
lothorax, frontal aspect (C). Scale bar = 1.5 cm. This figure is available in
colour at Journal of Crustacean Biology online.
brown-black, in contrast to the pearl-white colour of Potamonautes
mhlophe sp. nov. The absence of dentition on the anterolateral
margins of the carapace in P. mhlophe sp. nov. easily differentiate
it from the southern African species (P.  calcaratus Gordon, 1929,
P.  dentatus, P.  parvispina Stewart, 1997, P.  warren, and P.  unispinus),
which are characterised by dentition on the anterolateral margins.
Differentiating the larger riverine species P. perlatus, P. barnardi Phiri
730
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& Daniels, 2014, and P. barbari Phiri & Daniels, 2014 from the new
species is more difficult, apart from the differences in distribution
and the diagnostic colour in life because of similarities in the
overall morphology of the species, with only subtle differences in
the male gonopod 1 among the species.
Nomenclatural statement:  A life science identifier (LSID)
number was obtained for the new species: urn:lsid:zoobank.
org:pub:7E1DCF14-6746-4F51-8C2C-C794C2A6DA7C.
SUPPLEMENTARY MATERA L
Supplementary material is available at Journal of Crustacean Biology
online.
S1 Table. Substitution models and for each of the three
mtDNA loci (12S rRNA, 16S rRNA, and COI) for the phyloge-
netic analyses.
ACKNOWLEDGEMENTS
We thank the National Research Foundation (NRF), Foundational
Biodiversity Initiative Programme for financial support. Mr.
Theo Busschau is thanked for photographing live and preserved
crabs, mouthparts, and male genitalia. We also thank the Central
Analytical Facility, University of Stellenbosch for sequencing the
samples. I would like to thank the two anonymous reviewers and
the editor for improving the overall quality of the manuscript.
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