Cryptic and widespread: a recipe for taxonomic misidentification in a freshwater crab species (Decapoda: Potamonautidae: Potamonautes sidneyi) as evident from species delimitation methods Author Daniels, Savel R. Author Busschau, Theo Author Gullacksen, Graeme Author Marais, Hannes Author Gouws, Gavin Author Barnes, Aaron text Zoological Journal of the Linnean Society 2023 2022-11-02 197 4 1005 1033 http://dx.doi.org/10.1093/zoolinnean/zlac068 journal article 10.1093/zoolinnean/zlac068 0024-4082 7803581 16EA47FF-8FAD-4C4F-9DD9-6255A2B29C9C POTAMONAUTES VALLES SP. NOV. ( FIGS 2 , 4 , 8A–C , 9A, B , 10A–D; TABLE 4) Zoobank registration : urn: lsid: zoobank. org:act: 6C7215BD-74DA-4780-9589-375930C5981C . Holotype : Muilhuis section, Blyde River Canyon Nature Reserve , Northern Escarpment , 24° 40 ′ 14.8 ″ S , 30° 52 ′ 34.1 ″ E , 1320 m a.s.l. , Mpumalanga Province , South Africa , SAM MB-A 094482 , one male . Hand collected by H. Marais , 12 July 2021 . Collected in fastflowing mountain streams. Paratype : Muilhuis section, Blyde River Canyon Nature Reserve , Northern Escarpment , 24° 40 ′ 14.8 ″ S , 30° 52 ′ 29.2 ″ E , 1308 m a.s.l. , Mpumalanga Province , South Africa , SAM MB-A 094483 , one male . Hand collected by H. Marais , 12 July 2021 . Collected in fastflowing mountain streams . Figure 9. Potamonautes Ʋalles sp. nov. , male holotype (SAM-MB A 094482). A, major right cheliped; B, minor left cheliped. Scale bar represents 10 mm. Figure 8. Potamonautes Ʋalles sp. nov. , male holotype (CL = 30.90 mm) (SAM-MB A094482) from Muilhuis section, Blyde River Canyon Nature Reserve, Mpumalanga Province, South Africa. A, whole animal dorsal aspect; B, whole animal ventral aspect; C, cephalothorax, frontal aspect. Scale bar represents 10 mm. Additional material examined: Muilhuis section, Blyde River Canyon Nature Reserve , Northern Escarpment , 24° 40 ′ 14.8 ″ S , 30° 52 ′ 34.1 ″ E , 1320 m a.s.l., Mpumalanga Province , South Africa , SAM MB-A 094484 , one male . Collected by H. Marais , 25 November 2020 . Collected in a fast-flowing mountain stream. Unnamed locality, 30 km north-east of Mbabane 26° 12 ′ 24.1 ″ S , 31° 17 ′ 59.6 ″ E , 1200 m a.s.l., Hhohho Province, Eswatini, 1 February 2019 . One recently moulted male, SAM MB-A 094485 collected by Theo Busschau. Unnamed locality, 30 km northeast of Mbabane 26° 12 ′ 35.4 ″ S , 31° 17 ′ 07.2 ″ E , 1200 m a.s.l. , Hhohho Province, Eswatini , three males , five females , three juveniles , SAM MB-A 094486 collected 1 February 2019 by Theo Busschau. One adult male, SAM MB-A094580 from Katrinasrust trout farm, 25° 42 ′ 05 ″ S , 30° 30 ′ 38 ″ E , 1664 m , Mpumalanga Province , South Africa , collected 28 November 2021 by Graeme Gullacksen. One adult female, SAM MB-A094581 from Katrinasrust trout farm, 25° 42 ′ 05 ″ S , 30° 30 ′ 38 ″ E , 1664 m Mpumalanga Province , South Africa , collected 1 December 2021 by Graeme Gullacksen. One adult male, SAM MB-A094582 from Katrinasrust trout farm, 25° 42 ′ 05 ″ S , 30° 30 ′ 38 ″ E , 1664 m Mpumalanga Province , South Africa , collected 15 January 2022 by Graeme Gullacksen . Diagnosis: Carapace very flat ( CH /CL = 0.51) ( Table 4 ); 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 carapace margin granulated ( Fig. 8A–C ). Third maxilliped: ischium with distinct vertical sulcus ( Fig. 8C ); s3/s4 complete, V-shaped, deep, midpoint almost meeting anterior margin of sternopleonal cavity; margins of s4 low, not raised ( Fig. 8B ). Cheliped: dactylus (moveable finger) slim, highly arched, enclosing oval interspace, with three larger teeth interspersed by smaller teeth along length; propodus (fixed finger) with four larger teeth interspersed by smaller teeth along length ( Fig. 9A, B ); carpus inner margin distal tooth large, pointed, proximal tooth reduced to granules ( Fig. 8A ); medial inferior margin of merus lined with series of small granules terminating distally at small, low distal meral tooth, lateral inferior margin smooth. G1 terminal article: one-third length of 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 curving sharply upward ( Fig. 10A, B ). Description: Based on male holotype ( holotype CWW 40.24 mm , Table 4 ). Carapace anterolateral margins granulated; widest anteriorly, narrowest posteriorly (CWP/CL 0.42); arched ( CH /CL 0.51) ( Fig. 8C ); front broad, one-third CWW (FW/CWW 0.40); urogastric, cardiac grooves distinct, other grooves faint or missing; postfrontal crest complete, anterolateral margin posterior to epibranchial tooth granulated, 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 ( Fig. 8A–C ); branchiostegal wall vertical sulcus faint, meeting longitudinal sulcus, dividing branchiostegal wall into three parts, suborbital, dorsal pterygostomial regions granulated, hepatic region smooth; suborbital margin faintly granulated. Third maxilliped: filling entire buccal frame, except for respiratory openings; exopod with long flagellum, ischium with faint vertical groove ( Fig. 10D ). Epistomial tooth large, triangular, margins lined by large granules. 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 sternopleonal cavity; margins of s4 low, not raised. Cheliped: dactylus (moveable finger) slim, arched, enclosing oval interspace, with three larger teeth interspersed by smaller teeth along length; propodus (fixed finger) with four larger teeth interspersed by smaller teeth along length ( Fig. 9A, B ); carpus distal tooth large, pointed, proximal tooth small but distinct, followed by granule; both inferior margins of merus lined by series of 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 spinelike bristles along segment. Pleon: outline broadly triangular with straight margins. G1 terminal article: short (one-third length of subterminal segment), curving away from midline, 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 the dorsal side of suture marking junction between terminal, subterminal parts ( Fig. 10A, B ). G2: terminal article long, flagellum-like, 0.5 times length of subterminal segment ( Fig. 10C ). Figure 10. Potamonautes Ʋalles sp. nov. , male holotype (SAM-MB A 094482). A, left gonopod 1, anterior view; B, left gonopod 1 posterior view; C, left gonopod 2 anterior view; D, right third maxilliped. Scale bar represents 10 mm. Table 4. Potamonautes Ʋalles sp. nov. , measurements (in mm) of the holotype and the range of additional male and female specimens examined. An * indicates that the structure was missing

Variable	Abbreviation	Holotype	Males	Females
carapace length	CL	30.90	38.18–23.95	29.13–26.73
carapace width at widest point	CWW	40.24	50.91–31.07	38.92–34.82
carapace posterior margin	CWP	13.24	19.47–11.64	15.01–13.93
frontal width	FW	16.48	19.40–10.96	13.92–13.36
distance between postfrontal	PFCD	4.47	4.46–3.20	3.49–2.91
crest and anterior margin
carapace height	CH	15.86	17.32–16.83	14.08–13.05
major cheliped propodus length	MCPL	31.38	40.22–18.90	23.91–21.09
major cheliped dactylus length	MCDL	19.22	23.75–10.78	13.86–12.93
pereiopod 2, merus length	P2ML	18.44	*–11.75	14.70–13.81
pereiopod 2, merus width	P2MW	6.53	*– 4.94	6.44–5.90
pereiopod 5, merus length	P5ML	17.76	*–12.77	15.91–14.11
pereiopod 5, merus width	P5MW	6.44	*–5.18	6.10–5.92

Molecular diagnosis: 16 S rRNA GenBank accession numbers: OL 685399 – OL 685400, OL 685418 –OL 685422, OL 685439–OL 685441. COI GenBank numbers: OL660549–OL660551, OL660573–OL660577. Distribution: Known from the Blyde River Canyon Nature Reserve, Katrinasrust trout farm (in the vicinity of Mbombela) and Sterkspruit in the northeast of the Mpumalanga Province in South Africa , as well as 30 km north-west of Mbabane, Eswatini ( Fig. 1 ; Supporting Information, Fig. S4A, B ). Remarks: Potamonautes Ʋalles is sister to P. danielsi in our phylogenetic analyses ( Fig. 4 ). Geographically the two species are distinct: P. Ʋalles is confined to the north-eastern corners of southern Africa, while P. danielsi occurs from southern KwaZulu-Natal along the Indian Ocean coastal (IOCB) forest belt and the adjacent interior into the Eastern Cape Province of South Africa . Morphologically the two can also be distinguished. Potamonautes Ʋalles is flat ( CH / CL = 0.51) and large bodied (CWW = 40.24 mm ), while P. danielsi is also flat ( CH /CL = 0.49) but smaller bodied (CWW = 25.8 mm ) ( Peer et al. , 2017 ). The terminal segment of gonopod 1 is short and 0.21 times the length of subterminal segment ( Peer et al. , 2017 ). Potamonautes sidneyi s.s. , is flat ( CH /CL = 0.54) and large bodied (CWW = 47 mm ). When alive, P. Ʋalles has a chocolate-brown colour with a purple pair of chelipeds with black tips (Supporting Information, Fig. S4C ). In addition, two burrowing swamp forest dwelling species are in this clade, P. isimangaliso and P. liƲidus . Potamonautes isimangaliso is endemic to the False Bay region of the iSimangaliso Wetland Park in the north-eastern KwaZulu-Natal Province , while P. liƲidus is present along the IOCB forest belt in KwaZulu-Natal and the Eastern Cape provinces of South Africa ( Daniels et al. , 2020b ). Ecologically, both P. isimangaliso and P. liƲidus are distinct from P. Ʋalles , because these species burrow into soil in their respective habitats, while P. Ʋalles is a boulder-stream dwelling species. The cephalothorax of P. liƲidus is ovoid, the postfrontal crest is incomplete, the carapace is vaulted and the branchial regions are convex. Potamonautes liƲidus has a sharp but small exorbital tooth but lacks epibranchial teeth and the carapace is highly vaulted ( CH /CL = 0.64), indicative of a semiterrestrial mode of life ( Gouws et al. , 2001 ). The species is moderately large (CWW = 37 mm ) ( Gouws et al. , 2001 ). The chelipeds are highly arched and adapted for burrowing ( Gouws et al. , 2001 ). Potamonautes liƲidus has a silver-blue shine of its carapace and may also be red to dark orange in colour ( Gouws et al. , 2001 ). The terminal segment of gonopod 1 is 0.25 times the length of the subterminal segment ( Gouws et al ., 2001 ). Similarly, the cephalothrax in P. isimangaliso is also ovoid, lacks epibranchial teeth, the exorbital teeth are reduced, the carapace is highly vaulted ( CH / CL = 0.57) and the species lacks any dentition on the anterolateral carapace margins ( Peer et al. , 2015 ). Potamonautes isimangaliso varies in colour from light brown, maroon, purple or brown/black in colour while it is light orange between the joints ( Peer et al. , 2015 ). Potamonautes isimangaliso is large bodied (CWW = 55.1 mm ) and inhabits ephemeral pans in sand forest where it burrows into the soil to a depth of 2–50 cm ( Peer et al. , 2015 ). The terminal segment of gonopod 1 is short and 0.23 times the length of the subterminal segment ( Peer et al. , 2015 ). In the Mpumalanga Province of South Africa , five additional species are present, P. flaƲusjo , P. mariepskoppie , P. sidneyi s.s. , P. unispinus and M. calcaratus . These species can be easily distinguished from P. Ʋalles . Potamonautes flaƲusjo is a semi-terrestrial, burrowing species that lives in marsh (vlei) areas adjacent to small streams. In P. flaƲusjo the habitat is burrows in peat soils adjacent to streams. The species burrows straight down into the peat soil to a depth of near 1 m , but the depth of the burrow will be determined by the watertable depth. During the winter months, the species seals the burrow entrance with soil from the tunnel and sits in a small, water-filled chamber at the base of the burrow. The species generally comes to the surface after the first summer rainfalls (Daniels, pers. obs.). The chelipeds of P. flaƲusjo show limited adaptations for burrowing ( Daniels et al. , 2014 ). Potamonautes flaƲusjo is a large-bodied species (CWW = 58.42 mm ) ( Daniels et al. , 2014 ). The carapace of P. flaƲusjo is highly arched ( CH /CL = 0.48) and the anterolateral margins of the carapace is smooth ( Daniels et al. , 2014 ). In addition, P. flaƲusjo is sulphur yellow ventrally and has yellow spots on the dorsal carapace surface. The terminal segment of gonopod 1 is short and 0.24 times the length of the subterminal segment ( Daniels et al. , 2014 ). Potamonautes mariepskoppie is a narrow endemic species confined to the Mariepskop area of the Blyde River Canyon Nature Reserve where it occurs in swampy areas. The species has a modified cheliped, and the dactylus is large and shovel-shaped and adapted for burrowing into soft mud ( Daniels et al., 2021 ). Unpublished genetic data corroborate the presence of the species at Haenertsburg in the Limpopo Province (Daniels, unpublished). In P. Ʋalles , the dactylus and propodus are both highly arched, a pattern typically observed in stream-dwelling mountain crab species that live under rocks and boulders, and are not associated with a burrowing mode of life. In addition, in P. Ʋalles , the anterolateral carapace margin lacks any dentition. Potamonautes unispinus has a single tooth on the anterolateral carapace margin and is common and widespread in rivers (Stewart & Cook, 1997). Potamonautes Ʋalles can be differentiated from P. sidneyi s.s . by its highly arched dactylus and propodus, and its confinement to fastflowing mountain streams. Maritimonautes calcaratus is phylogenetically distinct from P. Ʋalles , with a near ovoid and arched carapace ( CH /CL = 0.51), and a thin spine-like tooth on the anterolateral carapace margin ( Reed & Cumberlidge, 2004 ; Cumberlidge & Daniels, 2022 ). In P. Ʋalles , the anterolateral carapace margin is granulated and it lacks a spine. The chelipeds (both propodus and dactylus) of M. calcaratus are flat and broad and adapted for digging (Daniels, pers. obs). Maritomonautes calcaratus inhabits ephemeral pans, where it burrows into the soft, sandy edges of the pans to a depth of 70 cm ( Daniels et al. , 2002 ). The terminal article of gonopod 1 in M. calcaratus is short in comparison with P. Ʋalles ( Cumberlidge & Daniels, 2022 ). The distribution of M. calcaratus in South Africa is restricted to the Mpumalanga Province where it is present in the Kruger National Park, and the species is also present in neighbouring Mozambique ( Reed & Cumberlidge, 2004 ; Cumberlidge & Daniels, 2008 ). The distribution range and habitat of M. calcaratus does not overlap with P. Ʋalles . Etymology: Named for its presence in valleys, hence the Latin word Ʋalles was used as a species epithet. It is a noun in apposition.