New endemic species of freshwater crayfish Parastacus Huxley , 1879 ( Crustacea : Decapoda : Parastacidae ) from the Atlantic forest in southern Brazil

Two new species of Parastacus Huxley, 1879 are described from the Atlantic Forest in southern Brazil: Parastacus buckupi sp. nov. from a highland stream in the hydrographic basin Maquiné river, Maquiné, state of Rio Grande do Sul; and Parastacus pilicarpus sp. nov. from a first order stream in the hydrographic basin Araranguá River, Morro Grande, state of Santa Catarina. Parastacus buckupi sp. nov. is distinguishable from all other Parastacus species via its large and globose chelipeds, by the presence of tufts of long and simple setae in the cutting edge of cheliped fingers, which are more abundant proximally and by possessing a telson with an acute distal margin. Parastacus pilicarpus sp. nov. is diagnosed mainly by the distal portion of the carpus which possess tufts of long, simple setae, and by having an epistome whose anteromedian lobe is heptagonal and as long as it is wide. Finally, P. pilicarpus antennal scales lateral margin terminates in a very strong terminal spine and mandibles incisive lobe has nine teeth, with the first tooth from the anterior margin orcid.org/0000-0002-4590-3049 orcid.org/0000-0002-8623-1576 orcid.org/0000-0002-7587-3936

The aim of this contribution is to describe two new species of Parastacus collected from first order streams in RS and SC, Brazil.The habitat characterization and the conservation status based on the IUCN Red List criteria are provided for both species.

Morphological analysis
The type material was deposited in the Museu de Zoologia of the Universidade de São Paulo (MZUSP), São Paulo, Brazil, and in the Carcinological Collection of the Departamento de Zoologia, Instituto de Biociências, Universidade Federal do Rio do Grande do Sul (UFRGS), Porto Alegre, Brazil.The comparative material is deposited in UFRGS.The drawings were made with the aid of a stereomicroscope fitted with a camara lucida.All measurements were performed with vernier caliper with 0.1 mm accuracy and a millimetric ocular on a stereomicroscope.Shape and size of the S2 pleura were defined according to Ribeiro et al. (2016).Sex was identified based on the morphology of the gonopores according to Rudolph (1997).Morphological descriptions are based on Riek (1971), Buckup and Rossi (1980), Hobbs (1987), Morgan (1997), Holdich (2002) and Ribeiro et al. (2016;2017).Branchial count follows Huxley (1879).The taxonomic classification follows Crandall and De Grave (2017).

Conservation analysis
The extinction risk of the new species was defined according to the B1 sub-criterion, sub-items "a" and "b" of the International Union for Conservation of Nature -IUCN (IUCN 2012).This sub-criterion takes into consideration the estimated Extent of Occurrence (EOO) which was calculated in Arcview 9.3 program (ESRI, 2009).The definition of the hydrographic basins follows the Otto Bacias shape method (level 4) (ANA, 2006).The definition of each measurement can be found in Ribeiro et al. (2016), with the exception of PL and PW following the definition of pleon by Schram (2013).Description of the holotype.Rostrum: triangular, longer than wide (RW 89% of RL), short (10% of CL), reaching proximal portion of the second article of the antennular peduncle (Fig. 1A-C).Dorsum straight, apex inverted U-shaped, ending in an upward blunt spine (Fig. 1B, C).Few plumose setae on lateral margins.Rostral carinae almost straight, prominent and narrow, extending back to carapace, surpassing rostral basis (Fig. 1B, C); sides convergent and basis slightly divergent (Fig. 1B).
Tailfan: telson weakly calcified in the distal margin, subrectangular, longer than wide (TeW 90% of TeL), with small sharp spines on lateral margins; acute distal margin with abundant long plumose setae and short simple setae; dorsal surface with tufts of short setae and inconspicuous dorsomedian longitudinal groove (Fig. 1G).Uropod protopod bilobed, with rounded and unarmed margins; proximal lobe largest (Fig. 1G); exopod lateral margin bears a small and sharp spine, mid-dorsal carina few prominent, ending in a sharp spine; transverse suture (diaeresis) straight, with four dorsolateral spines (outer) and four dorsolateral spines (inner) on right exopod and six dorsolateral spines (outer) and five dorsolateral spine (inner) on the left exopod; endopod, mid-dorsal carina few prominent, ending in a very sharp spine; lateral margin with one sharp spine at level of diaeresis (Fig. 1G).
Epistome: anterolateral section with a blunt conical projection.Posterolateral section smooth and with deep lateral grooves converging to the basis of the anteromedian lobe, and small median circular concavity.Anteromedian lobe pentagonal, as long as wide, apex acute and straight with serrate setae, reaching median part of antepenultimate article of antennal peduncle; dorsal surface straight, and basis with a shallow groove (Fig. 2A).
Thoracic sternites: SLP4 smallest and close to each other, median keel present and not inflated; SLP5 small and close to each other, median keel present and not inflated; SLP6 larger than SLP4 and SLP5 and with a slightly concave surface, median keel inflated; SLP7 largest and with surface slightly concave, median keel inflated, bullar lobes absent; SLP8 small and slightly concave, median keel absent, vertical arms of paired sternopleural bridges close to each other, bullar lobes separated and clearly visible (Fig. 2B, C).
Antennule: internal ventral border of basal article with one small sharp spine (Fig. 2A).
Antenna: when extended back reaching up to the posterior margin of the carapace.Antennal scale widest distally at midlength, reaching midlength of third antennal article, ASW 42.7% of ASL (Fig. 2A, D), lateral margin curved with strong spine and distal margin straight.Coxa with prominent carina above nephropore and a blunt spine laterally displaced.Basis unarmed (Fig. 2A).
Incisive lobe with ten teeth.The fourth tooth from the anterior margin is the largest (Fig. 2E).
Third maxilliped: ischium, ventral surface partially covered by tufts of long and simple setae (Fig. 2F); dorsal surface glabrous (Fig. 2G).Crista dentata bearing 27 and 30 teeth in right and left ischium respectively.Merus ventral surface partially covered by tufts of long and simples setae.Exopod longer than ischium,  with flagellum reaching proximal margin of merus (Fig. 2F, G).
Second pair of pereiopods: ventral and dorsal surface of carpus, propodus and dactylus with sparse covering of simple long setae (Fig. 2J).
Gonopores: Presence of both genital apertures on coxae of third and fifth pairs of pereiopods.Female gonopores semi-ellipsoidal (maximum diameter 1.46 mm) with a well-calcified membrane.Male gonopores rounded, opening onto apical end of a small, fixed, calcified and truncated phallic papilla, close to inner border of ventral surface of coxae of fifth pair of pereiopods.Male cuticle partition present (Fig. 2B).
Branchial count: 20 + epr + r.Branchial arrangement follows the same described by Huxley (1879) and Hobbs (1991) with the epipod of the first maxilliped with rudimentary podobranchial filaments.
Color of living specimens.Rostrum dark brown.Antennal flagellum light brown.Cephalothorax anterior and lateral regions dark brown to reddish brown.Chelipeds brown with dark brown fingers.Pereiopod pairs 2-5 light brown to dark brown.Dorsal pleon and tailfan light brown to reddish brown (Fig. 3C).
Remarks.Parastacus buckupi sp.nov. is morphologically similar to P. brasiliensis in having a wide pleon, large chelipeds, rostral apex inverted U-shaped with an upward blunt spine and a pentagonal anteromedian lobe of epistome.Parastacus buckupi sp.nov. is also similar to P. fluviatilis and P. pilimanus in having the cutting edge of fingers covered by tufts of long and simple setae (Fig. 7).Parastacus buckupi sp.nov.differs from all other Parastacus species by the large and globose chelipeds with tufts of plumose setae in the cutting edge of fingers, which are more abundant in the proximal portion (Fig. 7A) and telson subrectangular with acute distal margin (Fig. 1A, G).It was observed that the distal margin of the telson is rounded in smaller specimens (Paratype 1-5; Tab. 1).All specimens are intersexed.In males paratypes, the female gonopores are semi-ellipsoidal (average maximum diameter 1.76 mm) covered by a calcified membrane.The male gonopores are similar in all type material.
Habitat and Ecology.Carvão stream is included in the Serra Geral Biological Reserve (Vilella et al., 2004), with headwaters located about 800 m above sea level (a.s.l.; Fig. 3A, B).This stream is nearly 3.5 km of length with a catchment area of approximately 6 km² and the margins are surrounded by riparian vegetation including Araucaria angustifolia (Bertol.)Kuntze, 1898 and grasslands (Araucaria Forest province) (IBGE, 1990;Vilella et al., 2004;Sydow et al., 2008 2014).The substrate of the creek is basaltic with arenite in the lower portions (Vilella et al., 2004) (Fig. 3A, B).Data concerning burrow morphology and burrowing behavior are not available since they were not recorded when the specimens were collected.However, it is possible that the new species shows some level of burrowing activity especially considering that the burrowing behavior is probably found in all species of Parastacus (Buckup and Rossi, 1980).
Distribution.Parastacus buckupi sp.nov. is so far recorded in Carvão stream in the municipality of Maquiné, state of Rio Grande do Sul, southern Brazil (Fig. 8).
Conservation status.The EOO was estimated at approximately 301.885 km² (Fig. 8), indicating that this species can be included in the Endangered -EN category, in which the EOO is less than 5,000 km² (IUCN, 2012).The species is categorized as EN under the subitem "a": for an EOO, which is severely fragmented; and subitem "b" (iii): continuing decline in quality of habitat.Both subitems are compatible with the threats existing in the species occurrence area: a small fragment of a stream with approximately 6 km² of catchment area (Vilella et. al., 2004).The region of Maquiné basin is impacted mainly through the substitution of natural vegetation by agriculture (Becker et al., 2004).Conservation in the Maquiné basin is of fundamental importance since the region constitutes the southern limit of dense Ombrophylous Forest (Atlantic Forest) and all associated animals and plants (Bencke and Kindel, 1999;Becker et al., 2004).We suggest therefore that the conservation status of this species be classified as ENDANGERED B1ab(iii).Type material.Holotype: male, Brazil, Santa Catarina, Morro Grande, Bacia do Rio Araranguá (28°41'57.44"S49°48'8.81"W),20/VIII/2015, coll.C. Feltrin & F.H. Llanos (MZUSP 36516).Paratypes: 1 -Brazil, Santa Catarina: one female, same data as holotype (UFRGS 6303); 2A and 2B -two males, same data as holotype (UFRGS 6304); 3 -one female, same data as holotype (UFRGS 6305); 4 -one male, same data as holotype (UFRGS 6306); 5 -one juvenile, same data as holotype (UFRGS 6506).
Diagnosis.Wide front with a short triangular rostrum.Rostral apex inverted U-shaped and unarmed.Suborbital angle > 90°.Postorbital carinae weakly proeminet.Cervical groove weekly V-shaped.Areola wide, 2.5x as long as wide.Mandible with caudal process bicuspidate with one cephalodistal cusp and one small distoproximal cusp.Incisive lobe with nine teeth.The first tooth from the anterior margin is the largest.S2 pleurae low and short with shallow groove parallel to margin.Epistome with the anteromedian lobe heptagonal, as long as wide.Antennal scale lateral margin curved with a very strong terminal spine.Distal portion of the cheliped carpus with tufs of longs e simple setae.Chelipeds laterally flattened with the cutting edge of the fingers densely covered by setae.
Tailfan: telson weakly calcified in the distal portion, subrectangular, longer than wide (TeW 81.7% of TeL), with inconspicuous sharp spines on lateral margins; rounded distal margin with abundant long plumose setae and short simple setae.Dorsal surface with tufts of short setae and inconspicuous dorsomedian longitudinal groove (Fig. 4G).Uropod protopod bilobed, with rounded and unarmed margins; proximal lobe largest; exopod lateral margin bears a small and sharp spine, mid-dorsal carina few prominent, ending in a sharp spine; transverse suture (diaeresis) straight, with three dorsolateral spines (outer) and two dorsolateral spines (inner) on right exopod and two dorsolateral spines (outer) and three dorsolateral spine (inner) on the left exopod; endopod, mid-dorsal carina few prominent, ending in a very sharp spine; lateral margin with one sharp spine at level of diaeresis (Fig. 4G).
Epistome: anterolateral section with a sharp conical projection.Posterolateral section smooth and with deep lateral grooves converging to the basis of the anteromedian lobe, and large median circular concavity.Anteromedian lobe heptagonal, as long as wide, apex acute and straight, reaching the median part of antepenultimate article of antennal peduncle; dorsal surface straight, and basis with a shallow groove (Fig. 5A).
Thoracic sternites: SLP4 smallest and close to each other, median keel present and not inflated; SLP5 small and close to each other, median keel present and not inflated; SLP6 larger than SLP4 and SLP5, separated to each other and with a slightly concave surface, median keel not inflated; SLP7 largest, close to each other and with surface slightly concave, median keel not inflated, bullar lobes absent; SLP8 larger than SLP4, SLP5 and SLP6 and with a slightly concave, median keel absent, vertical arms of paired sternopleural bridges close to each other, bullar lobes close to each other and clearly visible (Fig. 5B, C).
Antennule: internal ventral border of basal article with small sharp spine (Fig. 5A).
Antenna: when extended back reaching up to the posterior margin of the carapace; antennal scale widest distal at midlength, almost reaching the end of third antennal article, ASW 59% of ASL (Fig. 5A, D), lateral margin curved with a very strong terminal spine and distal margin emarginated.Coxa with prominent carina and blunt spine above nephropore.Basis unarmed (Fig. 5A).
Mandible: cephalic molar process molariform, caudal molar process bicuspidate with one cephalodistal cusp and one small distoproximal cusp.Incisive lobe with nine teeth.The first tooth from the anterior margin is the largest (Fig. 5E).
Second pair of pereiopods: ventral and dorsal surface of carpus, propodus and dactylus with sparse covering of simple and long setae (Fig. 5J).
Gonopores: Presence of both genital apertures on coxae of third and fifth pairs of pereiopods.Female gonopores semi-ellipsoidal (maximum diameter 1.69 mm) with a well-calcified membrane.Male gonopores rounded, opening onto apical end of a small, fixed, calcified and truncated phallic papilla, close to inner border of ventral surface of coxae of fifth pair of pereiopods.Male cuticle partition present (Fig. 5B).
Branchial count: 20 + epr + r.Branchial arrangement follows the same described by Huxley (1879) and Hobbs (1991) with the epipod of the first maxilliped with rudimentary podobranchial filaments.
Color of living specimens.Rostrum dark brown.Antennal flagellum light brown.Cephalothorax anterior and lateral regions dark reddish brown to light reddish brown.First pair of pereiopods dark reddish brown dorsally and reddish brown in ventral and lateral surfaces.Dactylus dark reddish brown in the proximal portion and reddish brown distally.Pereiopod pairs 2-5 light reddish brown to reddish brown.Dorsal pleon and tailfan reddish brown (Fig. 6C).Remarks.Parastacus pilicarpus sp.nov. is morphologically similar to P. buckupi sp.nov. in having the rostral apex inverted U-shaped and short rostral carinae.Parastacus pilicarpus sp.nov. is also similar to P. buckupi sp.nov., P. fluviatilis and P. pilimanus by the presence of tufts of long and simple setae on the cutting edge of fingers of chelipeds (Fig. 7).Parastacus pilicarpus sp.nov.differs from all other Parastacus spp. in having the epistome anteromedian lobe heptagonal, as long as wide (Fig 5A ), antennal scale lateral margin curved with a very strong terminal spine (Fig. 5D), mandible incisive lobe with the first tooth from the anterior margin being the largest and the presence of tufts of long and simple setae in the distal portion of carpus (Fig. 5I).All specimens are intersexed.In males paratypes the female gonopores are semi-ellipsoidal (average maximum diameter 1.69 mm) covered by a calcified membrane (Fig. 5B).The male gonopores are similar in all type material.
Habitat and Ecology.All animals were collected in the headwaters of Manoel Alves stream which is considered a first order stream.Based on photographs (Fig. 6A, B), it is possible to infer that the margins are surrounded by riparian vegetation, gravel and rocks probably from basaltic origin.Despite the absence of information on burrowing behavior and burrow morphology, the possibility of the species shows some burrowing ability, cannot be discarded since according to Buckup and Rossi (1980) burrowing behavior is probably found in all species of Parastacus.

Figure 3 .
Figure 3. Parastacus buckupi sp.nov, habitat and living specimens.A, B, Typical habitat, a first order stream at Maquiné, state of Rio Grande do Sul; C, living specimen, habitus, dorsal view.Photographs by Fábio S. Vilella.No available information of scale in photograph C.

Figure 8 .
Figure 8. Distribution of Parastacus buckupi sp.nov.(star) and P. pilicarpus sp.nov.(triangle) in the states of Rio Grande do Sul and Santa Catarina, southern Brazil.

Table 1 .
Measurements (mm)of the type series of Parastacus buckupi sp.nov.For abbreviations see Material and Methods section.

Table 2 .
Measurements (mm) of the type series of Parastacus pilicarpus sp.nov.(* broken appendage).For abbreviations see Material and Methods section.