The hole is deeper: description of two new species within the Parastacus brasiliensis (von Martens, 1869) species complex with an integrative taxonomy approach Author Huber, Augusto Frederico 0000-0002-4590-3049 gutofh @ yahoo. com. br; https: // orcid. org / 0000 - 0002 - 4590 - 3049 gutofh@yahoo.com.br Author Araujo, Paula Beatriz 0000-0002-7587-3936 pabearaujo @ gmail. com; https: // orcid. org / 0000 - 0002 - 7587 - 3936 pabearaujo@gmail.com Author Ribeiro, Felipe Bezerra 0000-0002-8623-1576 Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo (USP), Ribeirão Preto, São Paulo, Brazil & fbribeiro. bio @ gmail. com; https: // orcid. org / 0000 - 0002 - 8623 - 1576 fbribeiro.bio@gmail.com text Zootaxa 2022 2022-07-22 5168 3 251 284 journal article 105314 10.11646/zootaxa.5168.3.1 261f63be-395f-4609-9fa7-96c6c05fb7d4 1175-5326 6882776 3624B353-61FF-4A2E-8583-453822F49948 Parastacus guapo sp. nov. Huber, Araujo & Ribeiro ( Figs. 1–6 ) Type series. Holotype : adult male , Brazil , Rio Grande do Sul , Pantano Grande , Horto Sanga das Pedras , Zona Alagada ( 30°13’28.4”S , 52°24’44.1”W ) 20/X/2020 , coll. A.F. Huber ; K.M. Gomes & F.B. Ribeiro ( MNRJcarcino 030200 ). Paratype 1: 1 female , same data as holotype (MNRJcarcino 030201); Paratype 3: 1 female , same data as holotype (MNRJcarcino 030202); Paratypes 2, 4–6: 1 male , 3 females and 2 juveniles , same data as holotype ( UFRGS 6932). FIGURE 1. Distribution of Parastacus guapo sp. nov. and Parastacus gomesae sp. nov. in the state of Rio Grande do Sul, southern Brazil. RS—state of Rio Grande do Sul, SC—state of Santa Catarina. Etymology. Named based on the regional expression “guapo”, originally of spanish language and a typical expression in the state of Rio Grande do Sul , which means beautiful, charming and elegant. It refers to the greenish blue color present principally in chelipeds of this species. We also suggest the common name “guapo crayfish” for this species. Diagnosis. Front with short triangular rostrum. Rostral apex shaped as inverted “U”, with long straight up-ward blunt spine. Eyes small. Suborbital angle>90° and unarmed. Postorbital carinae more prominent in anterior and middle portions. Cervical groove weakly V-shaped. Areola narrow and barely discernible. Telson subrectangular with large sharp spines on lateral margins. Epistome anteromedian lobe pentagonal and anterolateral section with big conical projection ending in a big sharp spine on both sides. Mandible with caudal molar process bicuspidate with one cephalodistal cusp and one medium conical distoproximal cusp; incisive lobe with nine teeth (the third tooth from the anterior margin is the largest). Internal surface of chelipeds palm with sparse tubercules. Description of holotype . Rostrum : triangular, longer than wide (RW 84.6% of RL), short (14.2% of CL), reaching distal portion of second article of antennular peduncle ( Fig. 2A–C ). Dorsum straight curved downward in anterior portion, apex inverted “U”-shaped, ending in a long straight upward blunt spine ( Fig. 2B, C ). Few plumose setae on lateral margins. Carinae straight, prominent, narrow, extending back to carapace and long, surpassing rostral basis; rostral carinae sides slightly convergent and rostral carinae basis parallel ( Fig. 2B, C ). Cephalon : Carapace lacking spines but with some tubercles above the anterior region of the cervical groove ( Fig. 2C ). CeL 67.1% of CL. Eyes small (CMW 60% of OW); suborbital angle>90°, unarmed ( Fig. 2C ). Front narrow (FW 45.3% of CW). Postorbital carinae little longer than rostral carinae (RCL 87.1% of POCL) and more prominent in anterior and middle portions, more discreet in final portion. Lateral cephalic edge with moderate setation ( Fig. 2A–C ). Thorax: carapace laterally compressed, deep and narrow (CD 50.7% of CL; CW 46% of CL). Cervical groove weakly V-shaped. Branchiocardiac grooves inconspicuous ( Fig. 2A ). Areola narrow, 2.5x as long as wide (AreL 28.5% of CL) ( Fig. 2A ). Pleon: lacking spines or tubercles, short and wide (PL 71% of CL; PW 82% of CW), smooth, covered with small setae on pleural margins ( Fig. 2A ). Pleural somites with rounded posterior margins. S1 pleurae with a small distal lobe slightly overlapped by S2 pleurae. S2 pleurae low and short with shallow groove parallel to margin and a deep groove above ( Fig. 2E ). Tailfan : telson a little less calcified in the distal margin, subrectangular, longer than wide (TeW 84.9% of TeL), with large and sharp spines on lateral margins; rounded distal margin with abundant long plumose setae and short simple setae. Dorsal surface with sparse tufts of short setae and inconspicuous dorsomedian longitudinal groove ( Fig. 2A, G ). Uropod protopod bilobed, with rounded and unarmed margins and some simple setae in the margin; proximal lobe largest. Exopod lateral margin bears a sharp spine, mid-dorsal carina prominent ending in a sharp spine. Transverse suture (diaeresis) straight with eight dorsolateral spines (outer) and six dorsolateral spines (inner) on both exopods. Endopod, mid-dorsal carina prominent, ending in a big sharp spine; lateral margin with one sharp spine at level of exopod transverse suture ( Fig. 2G ). Epistome: anterolateral section with big conical projection ending in a big sharp spine on both sides, with three and four small tubercles between the apex and basis of the conical projection in the left and right sides respectively, and a small circular median concavity. Posterolateral section smooth and with lateral grooves converging to the basis of the anteromedian lobe. Median section with a longitudinal groove deeper close to the anteromedian lobe. Anteromedian lobe pentagonal, apex rounded by use, a little wider than long, with some sparse serrated setae, reaching median part of first article of antennal peduncle; dorsal surface concave with a groove from the middle of the lateral sides to the apex and basis with a shallow groove ( Fig. 3A ). Thoracic sternites: SLP4 small and close to each other, median keel present and not inflated; SLP5 same size of SPL4 and close to each other, median keel present and not inflated; SLP6 larger than SLP4, SLP5 and similar size to SLP8 with a slightly concave surface, median keel present and not inflated; SLP7 largest and with surface slightly concave, median keel inflated, bullar lobes absent; SLP8 median size with and slightly concave, median keel absent, vertical arms of paired sternopleural bridges separated to each other, bullar lobes absent ( Fig. 3B, C ). Antennule : internal ventral border of basal article without spines ( Fig. 3A ). Antenna : when extended back reaching the posterior margin of SIII.Antennal scale widest at midlength, reaching the middle of the third antennal article, ASW 43.3% of ASL ( Fig. 3A, D ), lateral margin straight and distal spine sharp and well developed. Coxa with weakly prominent carina above nephropore and three blunt spines different in size laterally distributed. Basis unarmed ( Fig. 3A ). Mandible: cephalic molar process molariform, caudal molar process bicuspidate with one bigger cephalodistal cusp and one medium conical distoproximal cusp. Incisive lobe with nine teeth. The third tooth from the anterior margin is the largest ( Fig. 3E ). Third maxilliped: ischium with some tubercles with tufts of short and composed setae on the middle portion and tufts of long simple setae on inner margin of ventral surface ( Fig. 3F, G ); dorsal surface with some sparse simple setae close to the outer margin ( Fig. 3G ); crista dentata bearing 24 teeth in the right and 21 in the left ischium ( Fig. 3F, G ). Merum, dorsal surface glabrous. Merum ventral surface sparsely covered by long smooth simple setae in the median region and with serrated setae on the distal of inner margin ( Fig. 3H ); Exopod longer than ischium, with flagellum reaching the initial ⅓ of merum and with tufts of long and composed setae in the last articles and tufts of composed setae in the basal article ( Fig. 3F, G ). First pair of pereiopods (chelipeds) : large and subequal, laterally flattened (RPrT 24.5% of RPrL; LPrT 24.7% of LPrL) ( Fig. 2A ; Fig. 3H, I ). Ischium ventral surface with 28 tubercles on both sides. Merus: right merus (RML) 56.7% of propodus length (RPrL); left merus (LML) 56.9% of propodus length (LPrL); ventral surface with two longitudinal series of tubercles: inner series with 21 tubercles, outer 24, plus 53 mesial tubercles irregularly distributed on right merus; inner series with 21 tubercles, outer 24, plus 55 mesial tubercles irregularly distributed on left merus; dorsal and midventral spines medium sizes and blunts. Carpus with dorsomedial surface with a shallow longitudinally groove and the distal margin with short and simple setae ( Fig. 2A ; Fig. 3H, I ). Internal dorsolateral margin with row of tubercles, increasing in size distally; inner surface with 37 and 33 small mesial tubercles in right and left sizes respectively. Carpal spine present and small. Propodus moderate (RPrW and LPrW) 43.5% of length in right cheliped and 42.6% in left cheliped. Dorsal surface of palm with 2–3 rows of verrucous tubercles irregularly distributed ( Fig. 3H, I ). Inner margin with sparse tubercles more concentrated in the low portion and close to the insertion of dactylus. Ventral surface bearing 2-3 irregularly rows of squamose tubercles, surpassing the beginning of the fixed finger ( Fig. 3H ). Dactylus: moving subvertically, right dactylus (RDL) 63.1% of propodus length (RPrL), left dactylus (LDL) 60.3% of left propodus (LPrL); dorsal surface with squamose tubercles decreasing in size from the basis to the apex of dactylus ( Fig. 3I ). Cutting edge of fingers visible. Fixed finger with thirteen teeth, sixth the largest, and twelve teeth, fourth the largest, on right and left sides respectively. Dactylus with sixteen and fourteen teeth in the right and left sides respectively, with the third teeth largest on both sides ( Fig. 3H, I ). Second pair of pereiopods : dorsal surface of dactylus and ventral surface of propodus with tufts of short and long simple setae, dorsal surface of propodus with long and simple setae. Dorsal surface of carpus with long composed setae ( Fig. 3J ). Gonopores : Presence of both genital apertures on coxae of third and fifth pairs of pereiopods. Female gonopores semi-ellipsoidal (maximum diameter 1.43 mm ) with well-calcified membrane. Male gonopores rounded, opening onto the apical end of a small, fixed, calcified and truncated phallic papilla, close to the inner border of the ventral surface of coxae of the fifth pair of pereiopods. Male cuticle partition present ( Fig. 3B ). 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. Measurements . Holotype male, CL 31.06 mm and TL 64.47 mm . In type series, CL ranging from 19.85 to 31.06 mm (23.1 ± 3.8 mm ). FW/CW: 0.45 ± 0.02 (min: 0.42; max: 0.47). RW/RL: 0.81 ± 0.07 (min: 0.70; max: 0.90). CMW/OW: 0.73 ± 0.07 (min: 0.59; max: 0.81). Postorbital carina longer or equal to rostral carina in all specimens analyzed. CW/PW: 1.11 ± 0.08 (min: 1.01; max: 1.22). AreW/RW: 0.89 ± 0.07 (min: 0.76; max: 0.96) ( Table 1 ). TABLE 1. Measurements (mm) of the type series of Parastacus guapo sp. nov. . For abbreviations, see Material and Methods.

Holotype	Paratype 1	Paratype 2	Paratype 3	Paratype 4	Paratype 5	Paratype 6
(MNRJ- carcino 030200)	(MNRJ- carcino 030201)	(UFRGS 6932)	(MNRJ- carcino 030202)	(UFRGS 6932)	(UFRGS 6932)	(UFRGS 6932)
Sex	M	F	M	F	F	F	F
TL	64.47	44.16	47.98	45.99	50.39	41.59	42.50
CL	31.06	20.92	23.20	21.82	24.11	19.85	20.81
CW	14.30	9.48	10.50	10.17	10.50	9.00	9.47
CD	15.74	11.24	12.27	11.56	12.43	10.11	11.07
CeL	20.85	14.13	16.01	14.33	16.31	12.70	13.42
RL	4.43	3.42	3.64	3.22	3.26	3.29	2.79
RW	3.75	2.84	2.85	2.33	2.95	2.31	2.38
RCL	5.94	4.84	5.72	5.10	5.25	4.35	4.60
CMW	1.54	1.29	1.30	1.26	1.47	1.30	1.26
OW	2.57	1.78	1.75	1.76	1.85	1.59	1.67
POCL	6.82	5.00	5.66	4.74	5.60	4.55	4.77
FW	6.48	4.51	4.92	4.54	4.78	3.77	3.95
ASL	4.64	2.83	3.71	2.74	3.30	2.97	2.68
ASW	2.01	1.59	1.68	1.31	1.60	1.30	1.31
AreL	8.86	5.87	6.10	6.49	6.91	5.54	5.65
AreW	3.47	2.18	2.42	2.24	2.61	2.11	2.28
LPrT	6.38	2.73	4.69	4.51	3.26	2.36	3.64
LPrL	25.77	11.25	16.02	15.11	13.13	9.81	12.99
LPrW	10.98	4.88	7.74	7.46	5.78	4.32	6.21
LDL	15.54	7.39	8.59	8.95	8.32	6.13	8.26
LML	14.66	8.04	9.02	9.54	8.79	7.21	7.85
RPrT	6.23	4.15	3.39	2.31	-	2.96	3.51
RPrL	25.39	14.37	13.59	11.34	-	10.58	13.34
RPrW	11.06	7.00	6.28	4.73	-	5.07	6.32
RDL	16.02	8.82	8.55	7.55	-	6.55	8.25
RML	14.40	8.69	8.86	7.70	-	7.12	7.74
PL	22.04	14.60	19.23	15.15	17.94	15.94	16.42
PW	11.73	8.94	10.31	9.02	9.54	8.68	7.73
TeL	9.40	6.32	6.95	6.60	7.58	6.19	6.03
TeW	7.98	5.45	6.30	5.78	6.49	5.08	5.39

FIGURE 2. Parastacus guapo sp. nov. . A, habitus dorsal view (holotype); B, cephalon dorsal view (holotype); C, cephalon lateral view (holotype); D, female pleonal somites dorsal view (paratype 1); E, male first, second and third pleonal pleura (holotype); F, female first, second and third pleonal pleura (paratype 1); G, telson and uropods dorsal view (holotype). Scale bars: A—1 cm; B, C, E and G—5.00 mm; F—3.33 mm. FIGURE 3. Parastacus guapo sp. nov. Holotype and paratypes. A, epistome (holotype); B, thoracic sternites and gonopores (holotype); C, thoracomere 8, caudal view (holotype); D, antennal scale lateral view (paratype 2); E, mandible (paratype 2); F, third maxilliped ventral view (paratype 2); G, third maxilliped dorsal view (paratype 2); H, first pereiopod lateral view (holotype); I, first pereiopod dorsal view (holotype); J, second pereiopod lateral view (holotype). Scale bars: A, C and J—3.33 mm; B, H and I—5 mm; D and E—1.6 mm; F and G—2.0 mm. FIGURE 4. Parastacus guapo sp. nov. Living specimens (holotype). A, habitus dorsal view; B, living specimen in the habitat; C, living specimen placed in an aquarium. CL—31.06 mm. Color of living specimens. Rostrum greenish blue to greenish-brown. Cephalothorax anterior and lateral regions brown with shades of light brown to greenish-blue. First pair of pereiopods greenish-blue on the external surface, greenish-blue with shades of light brown in internal surface and apex of dactylus and propodus orange-red to reddish-brown. Pereiopod pairs 2–5 light brown to light greenish-blue in the dorsal and lateral regions and light brown in the ventral region. Dorsal pleon and taifan brown with shades of light greenish-brown, low portions of lateral margins of pleon with shades of greenish-blue ( Fig. 4 ). Variations in paratypes . All paratypes present both masculine and feminine gonopores in the same individual. Male paratypes also present female gonopores semi-ellipsoidal (average maximum diameter 1.2 mm ) covered by a calcified membrane. Male gonopores are very similar in all male paratypes . Front wide in paratype 1 ( FW / CW = 47.5%). Rostrum of paratypes 3 and 5 long once they reach the distal portion of the third article of antennule. Suborbital angle 90° in paratype 5. Rostral and postorbital carena of similar sizes in paratypes 2 and 3. Pleon long in paratypes 2 and 5 ( PL / CL = 82.9% and 80.3%, respectively). S2 pleura moderate in paratypes 1 and 4, long in paratype 6, high and moderate in paratype 3 and high and long in paratype 5. Number of spines in exopod of tailfan varies between eight or nine dorsolateral spines (outer) and four, six or seven in dorsolateral spines (inner) on right side and eight or nine dorsolateral spines (outer) and five or six dorsolateral spines (inner) on the left side. Epistome anteromedian lobe longer than wide in paratype 2. Antennule internal ventral border of the basal article with one spine in the middle portion of left side in paratype 1 and with spines on both sides in paratypes 2, 4, 5 and 6. Antenna when extended back reaching the posterior margin of fourth pleura in paratypes 2 and 6. Antennal scale reaches the proximal margin of the third antennal article of antennule in paratype 1. Incisive lobe of the mandible has eight teeth in paratypes 6. The number of teeth in the crista dentata ranges from 21—27 in the left ischium and from 20—32 in the right ischium of the third maxilliped in the paratypes . Merus midventral spine doubles in paratype 5. Carpal spine more prominent and sharp in paratypes 1, 3 and 4. Dorsal spine sharp in both merus of chelipeds in paratypes 2 and 3. The number of teeth in fixed fingers of chelipeds and dactylus have a wide range of variation in size and number, which is probably related to the smaller size of paratypes when compared to holotype or regenerated chelipeds. Remarks. Parastacus guapo sp. nov. is morphologically similar to P. brasiliensis , P. buckupi , P. macanudo and P. promatensis in the general shape of rostrum, rostral carina length, shape of telson (subrectangular) and epistome anteromedian lobe pentagonal ( Fig. 2A–C, G ; 3A ; Table 2 ). However, it differs from: P. brasiliensis in rostral surface straight, sub-orbital angle> 90°, postorbital carinae prominent in anterior and middle portions, carpal spine present, internal surface of chelipeds palm with sparse tubercules, areola narrow and pleon short ( Fig. 2A–C ; 3A, H, I ; Table 2 ); P. buckupi in the same characters that P. brasiliensis , except the areola narrow, and is also different for mandible incisive lobe with nine teeth (the third tooth from the anterior margin is the largest), mandible caudal molar process bicuspidate, internal surface of chelipeds palm with sparse tubercules and telson with large lateral spines ( Fig. 2A–C, G ; 3E, H, J ; Table 2 ); P. macanudo in a bigger rostral spine, postorbital carinae prominent in anterior and middle portions, mandible incisive lobe with the third tooth the largest, 2–3 rows of verrucose tubercles irregularly distributed in the palm dorsal surface of chelipeds, internal surface of chelipeds palm with sparse tubercules and the habitat ( Fig. 2A–C ; 3E, H, J ; Table 2 ); P. promatensis in the apex of rostrum rounded, sub-orbital angle> 90°, postorbital carinae prominent in anterior and middle portions, cervical groove “V” shaped, carpal spine present, 2–3 rows of verrucose tubercles irregularly distributed in the palm dorsal surface of chelipeds and pleon short; however both species have internal surface of chelipeds palm with sparse tubercules ( Fig. 2 A-D; 3H; Table 2 ). Parastacus guapo sp. nov. differs from all other Parastacus species in having the epistome anterolateral section with large conical projection ending in a large sharp spine on both sides ( Fig. 3A ). Phylogenetic position. The phylogenetic analysis suggests, with high values of reliability (bootstrap and posterior probability), that P. guapo sp. nov. is a distinct species of P. brasiliensis s.s. , Lineages A–E, and other species of the genus included ( Fig. 5 ). The genetic distances estimated between P. guapo sp. nov. and P. brasiliensis s. s. was 5.35% (Sup. Table. 2 ). Besides that, the lineages range from 3.41% (Lineage C) to 5.95% (Lineage E), and of other species from 4.05% ( P. buckupi ) to 5.90% ( P. tuerkayi ) (Sup. Table. 2 ). Habitat and ecology. Parastacus guapo sp. nov. was collected in a wetland area, near a small stream, inside the Sanga das Pedras project of CMPC ( Fig. 1 ). This forestry area is located in the Baixo Jacuí hydrographic basin in the central region of the state of Rio Grande do Sul ( Fig. 6 ). The area is part of the physiographic region of Depressão Central, almost in the division with the Planalto Sul Rio Grandense region, and of the Pampa biome ( IBGE 2004a ; 2004b ; SIGBio-RS 2021). TABLE 2. Main characters distinguishing Parastacus guapo sp. nov. and Parastacus gomesae sp. nov. from other species of Parastacus cited in the Remarks section. Asterisks: one (*)—indicate variations in the type series, more information in Remarks section; two (**)—according to comparative material. For abbreviations, see Material and Methods.

Characteres	P. brasiliensis	P. buckupi	P. guapo sp. nov.	P. gomesae sp. nov.	P. macanudo	P. promatensis
Rostrum
Shape	Triangular or spatu- lated	Triangular	Triangular	Triangular	Triangular	Subtriangular
Apex	Rounded, ending in an upward spine	Rounded, ending in an upward spine	Rounded, ending in an upward spine	Rounded and smooth*	Rounded, ending in a tiny straight blunt spine*	Acute, ending in a short and depressed spine.
Dorsal Surface	Concave	Straight	Straight	Straight with a slightly concave in the anterior portion	Straight	Flat
Suborbital angle	(=90°)	(=90°)	>90°*	>90° *	>90°	(=90°)
Postorbital Carina	Weakly prominent	Weakly prominent	Prominent in anterior and middle portions	Prominent in anterior and middle portions	Weakly prominent	Prominent
Cervical groove	Weakly V-shaped	Weakly V-shaped	Weakly V-shaped	Weakly V-shaped	Weakly V-shaped	U-shaped
Epistome
Anteromedian lobe	Pentagonal	Pentagonal	Pentagonal	Heptagonal	Pentagonal	Pentagonal
Mandible
Incisive process	Nine teeth (the third tooth from the anterior margin is the largest)	Ten teeth (the fourth tooth from the anterior margin is the largest)	Nine teeth (the third tooth from the anterior margin is the largest)*	Seven teeth (the second tooth from the anterior margin is the largest)	Nine teeth (the second tooth from the anterior margin is the largest)	Nine teeth (the third tooth from the anterior margin is the largest)
Caudal molar process	Bicuspidate	Unicuspidate	Bicuspidate	Bicuspidate	Bicuspidate	Bicuspidate
Chelipeds
Carpal spine	Absent	Absent	Present	Absent	Present	Absent
Size and shape	Large and flattened	Large and globose	Large and flattened	Large and flattened	Large and flattened	Large and flattened

......continued on the next page TABLE 2. (Continued)

Characteres	P. brasiliensis	P. buckupi	P. guapo sp. nov.	P. gomesae sp. nov.	P. macanudo	P. promatensis
Palm dorsal surface	With 2-3 rows of verru- cose tubercles irregu- larly distributed	With 2 rows of verru- cose tubercles irregu- larly distributed	With 2-3 rows of verru- cose tubercles irregu- larly distributed	With 3-4 rows of verru- cose tubercles irregu- larly distributed	With a single row of verrucose tubercles ir- regularly distributed	With 3-4 rows of verrucose tubercles irregularly distributed
Palm internal surface	Smooth	Smooth	Tubercules in medial and low portions	Few small tubercules	Smooth	Tubercules in medial portion
Areola	Wide	Narrow	Narrow*	Narrow	Narrow	Narrow
Pleon
Shape	Long and wide	Short and wide	Short and wide*	Short and wide*	Long and wide	Long and wide
Telson
Lateral spines	Large and sharp	Small and sharp	Large and sharp	Small and sharp	Large and sharp	Large and sharp
Habitat	Streams	High altitude streams	Streams margins and overflow zone	Streams and overflow zone	Swamp forest	High altitude streams
References	Buckup & Rossi (1980) ; Miranda et al. (2018) ; Ribeiro et al. (2020 ; 2021)	Huber et al. (2018) ; Ribeiro et al. (2020 ; 2021)	Present contribution	Present contribution	Huber et al. (2020) ; Ribeiro et al. (2020 ; 2021)	Fontoura & Conter (2008) ; Miranda et al. (2018) ; Ribeiro et al. (2020)

FIGURE 5. Maximum likelihood analysis estimates phylogenetic relationships among selected Parastacus species based on 16S rRNA gene data from the mitochondrial genome. A, Cladogram showing the relation of analyzed sequences. B, Simplified phylogram with the lineages A–E, new species and other selected species of the genus. Numbers at nodes represent bootstraps/ posterior probabilities (ML/BAY) expressed as a percentage, values under 50% are omitted. New sequences are denoted by asterisks (*). For more information see Supplemental Table 1. Vegetation . The region has as predominant vegetation grassy-woody steppe with gallery forests or riparian vegetation associated with streams and their overflow zones ( Fig 6 A, B ). In addition to the presence of forestry areas of Eucalyptus sp. , the native plant formations of the region are mainly composed of grasses and bushes of the families Poaceae ( Paspalum spp. L., Andropogon lateralis Nees —“Capim Caninha’’ and Mnesithea selloana (Hack.) de Koning & Sosef ), Asteraceae ( Baccharis ochracea Spreng. —“Vassoura do Campo”, Vernonanthura nudiflora (Less.) H.Rob. , Senecio selloi (Spreng.) DC and Senecio brasiliensis (Spreng.) Less. —“Maria Mole”), Cyperaceae ( Eleocharis spp. R.Br., Rhynchosia diversifolia Micheli. and Cyperus spp. L.), Fabaceae ( Desmodium incanum (Sw.) DC —“Pega Pega”, Trifolium polymorphum Poir. —“Trevo” and Macroptilium prostratum (Benth.) Urb. ), Solanaceae ( Solanum spp. L.) and some small trees like Myrtaceae ( Psidium cattleianum Sabine —“Araçá”) ( Caumo et al. 2020 ) ( Fig. 6 ). FIGURE 6. Parastacus guapo sp. nov. Habitat and burrows. A, wetland area; B, small stream near the wetland area; C and D, chimneys indicated by the white arrows. Soil. The soil is classified as Dystrophic Red Argisols, but mixed with Dystrophic Red-Yellow Argisols ( Santos et al. 2011 ). These types of soils have accentuated red colors due to higher levels of iron oxides and usually occur in areas of undulating relief, but they can be identified in less steep areas. They have low fertility that could be explained by relief slope, nutrient limitations, strong acidity, high drainage capacity and also high susceptibility to erosion and degradation (AGEITEC 2021a; Santos et al. 2011 ; 2021a ; 2021b ; Streck et al. 2002 ; 2008 ). Burrowing behavior and burrow structure . According to observations of burrows structure and characteristics of the habitat, Parastacus guapo sp. nov. can be classified as a primary burrower with types 1a, b burrows, due to the construction of complex tunnels close and with direct and indirect connections to permanent water bodies ( Hobbs 1942 ; Horwitz & Richardson 1986 ; Richardson, 2007 ) ( Fig. 6 ). This species builds burrow galleries of 1–4 interconnected tunnels, which can reach a depth up to 1m ending in a life chamber. The entries of the galleries are delimited by simple holes or by chimneys, which can reach up to 8 cm high and wide ( Fig. 6C, D ). Distribution. Parastacus guapo sp. nov. appears to have limited distribution, being registered only in the municipality of Pantano Grande, central region of Rio Grande do Sul , southern Brazil ( Fig. 1 ). Conservation status. Data Deficient. The EOO and AOO were estimated as approximately 937 km ² and 1, 87 km ² (or 0, 38 km ² considering only preserved habitat), respectively ( Fig. 1 ). According to the sub-criteria B1 and B2, an EOO < 5,000 km ² and/or AOO < 500 km ², respectively, added to at least two conditions or subitems of this criterion categorizes the species in the threatened category Critically Endangered (CR) (IUCN 2019). It is important to consider that there is only one known location for this species and this is inside a large forestry area, which preserves only fragments of natural habitat. Therefore, it is probable that the subitem “a”, severely fragmented population or a low number of locations, and subitem “b” (iii): continuing decline in quality of habitat. However, due to the low number of surveys in the region and based on the current knowledge of this species, it can only be included in sub-item b (iii): continuous decline observed in habitat quality. Taking this into account, we classified this species as DATA DEFICIENT (DD).