Molecular phylogeny of the Austrolebias adloffi group (Cyprinodontiformes Rivulidae), with description of two new endangered and highly endemic species of annual killifishes from the Laguna dos Patos system, southern Brazil Volcan, Matheus Vieira Barbosa, Crislaine Robe, Lizandra Jaqueline Lanés, Luis Esteban Krause Zootaxa 2021 2021-04-27 4965 1 87 113 Volcan & Barbosa & Robe & Lanés, 2021 Volcan & Barbosa & Robe & Lanés 2021 [151,458,1601,1628] Actinopterygii Rivulidae Austrolebias Animalia Cyprinodontiformes 8 95 Chordata species lourenciano sp. nov.     Holotype. MCP 5440, male, 36.9 mmSL, Brazil, Rio Grande do Sul State, São Lourenço do Sulmunicipality, temporary pool in the floodplains of Arroio Pinto(tributary of Arroio São Lourenço), close to the BR-116 highway, Laguna dos Patossystem, 31°20’50”S 52°03’53”W, M. V. Volcan& Â.C. Gonçalves,  30 September 2013.    Paratypes. Allfrom Brazil, Rio Grande do Sul, São Lourenço do Sulmunicipality, Laguna dos Patossystem:  MCP54441, 5 males 24.9–32.3 mmSL (2 C&S), 6 females(3 C&S), 26.2–32.6 mmSL, same data as holotype;  MCP54442, 6 males25.1–33.0 mm SL, 6 females, 27.3–30.5 mmSL, same locality as holotype, M.  V. Volcan& Â.C. Gonçalves,  3 September 2013.  MCP54439, 1 male, 40.2 mmSL, 1 female, 39.7 mmSL, same locality of holotype, M.  V. Volcan, G. M. Wallwitz& J.  T. Basi,  16 August 2018.  MCP54443, 1 male, 36.9 mmSL, 3 females(1 C&S) 32.7–36.1 mmSL, temporary pool in the floodplains of Arroio Pinto, close to BR-116 highway, 31°20’48”S 52°03’48”W, M.  V. Volcan& Â.C. Gonçalves,  3 September 2013.   Diagnosis.The new species differs from all the other species of the genus, except  A. adloffi,  A. charrua,  A. cheffei, A. minuano,  A. nachtigalli,  A. nigrofasciatus,  A. reicherti,  A. pelotapesand  A. pongondoby the presence of a pair of black blotches vertically arranged on the posterior portion of the caudal peduncle in females (vs. absence of black blotches in the remaining species).  Austrolebias lourencianois distinguished from all other species of the  A. adloffispecies group by presenting a gray or yellowish green dorsal fin with light yellow or light bluish bars forming small triangles, interspersed with small dark brownish row at dorsal fin base (vs. never a similar pattern); by greenish blue anal fin with light iridescent stripes, limited to basal region of anal fin, sometimes as an extension of the transverse bars of the body (vs. never a similar pattern); and from all species of the  A. adloffispecies group, except  A. cheffei, A. pongondo, A. nachtigalli, A. nigrofasciatusand  A. reicherti, by presence of a black blotch on the distal portion of last anal and dorsal fin rays bordered anteriorly by a small light blue margin (vs. black blotch either absent or inconspicuous in the last anal and dorsal fin rays). Additionally,  A. lourencianodiffers from  A. adloffiby presenting posterior bars of body wider than interspace in males (vs. all bars narrower than interspace), greenish blue caudal fin, without dots, bars or spots on basal portion in males (vs. dark gray caudal fin, with light blue spots on basal portion), lower anal fin base length in males (38.1–43.8% SL, vs. 44.3–49.5% SL), by the position of dorsal fin origin in relation to the neural spines in males (8th–10th, vs. 7th–8th) and by lower counts of gill rakers in the first branchial arch (3+9–10, vs. 3–4+11).  Austrolebias lourencianodiffers from  A. charruaby lower number of vertebrae (27–28, vs. 29–31) and by the presence of a basihyal cartilage with about 60–70% of basihyal length (vs. 45–55%). Autrolebias lourencianodiffers from  A. cheffeiby the lower number of rays in the anal fin of males (21–24 vs. 25–27), by urogenital papilla not attached to anal fin in males (vs. urogenital papilla attached at base to 30% of its length to anal fin) and by dorsal-fin origin posterior to anal-fin origin in males (vs. dorsal-fin origin anterior to anal-fin origin). It differs from  A. minuanoby lower anal fin base length in males (38.1–43.8% SL, vs. 44.9–53.6% SL), lower head depth in females (95.7–105.3% HL, vs. 106.1–124.2% HL) and by lower number of teeth on second pharyngobranchial (1–3 vs. 4–7).  Austrolebias lourencianodiffers from  A. nachtigalliby the absence of golden to pink iridescence at distal half of dorsal fin in males (vs. presence of golden to pink iridescence at distal half of dorsal fin), by dark bluish gray caudal fin, without dots or spots in males (vs. light blue iridescence and faint elongated blue spots), by lower head width in males (53.1–65.2% HL, vs. 66.6–71.4% HL) and by lower head depth in females (95.7–105.3% HL, vs. 107.7–115.5 % HL). It differs from  A. nigrofasciatusby dorsal-fin origin posterior to anal-fin origin in males (vs. dorsal-fin origin anterior to anal-fin origin), by a greenish blue caudal fin, without dots or spots in males (vs. elongated light blue spots). It differs from  A. reichertiby higher dorsal fin base length in males (35.7–41.8% SL, vs. 34.2–34.9% SL).  Austrolebias lourencianodiffers from  A. pelotapesby having the urogenital papilla base free in males ( vs.urogenital papilla attached by a thin membrane to the anterior margin of the anal fin), by lower caudal peduncle depth in males (12.8–15.0% SL, vs. 15.1–17.3% SL), by lower pre-pelvic length (45.2–53.2% SL, vs. 53.9–60.7% SL) and lower anal fin base length (25.8–29.9% SL, vs. 30.4–36.1 % SL) in females; by basihyal cartilage with about 60–70% of basihyal length (vs. 40%), and by separated preopercular and mandibular series of neuromasts (vs. united mandibular and preopercular series). The new species differs from  A. pongondoby lower anal fin base length in males (38.1–43.8% SL, vs. 43.9–48.0% SL), lower head depth in females (58.6–71.3% HL, vs. 73.0–81.0% HL) and by basihyal cartilage with about 60–70% of basihyal length (vs. 40%).   Austrolebias lourencianois distinguished from all other species of the  A. adloffispecies group by one unique nucleotide substitution along the cytb alignment: cytb.457 (T> G) ( Fig. 2S). It is distinguished from  A.aff. minuano1 and from its sister clade (which contains  A.aff. minuano1 and  A. cheffei) by this and other two nucleotide substitutions: cytb.133 (A> C) and cytb.595 (C> T).  Genetic distance.From  A.aff. minuano1: 1.7–2.9%; from  A. cheffei: 1.9–2.6%; from  A.aff. minuano2: 3.1–4.0%; from  A. adloffi sensu lato:3.3–3.8%; from  A. pelotapes:3.6–4.5%; from  A. pongondo: 3.8–4.5%; from  A. bagual: 5.4–6.5%; from  A. nigrofasciatus: 6.9–9.2%; from  A. minuano sensustricto: 9.6–10.6%; and from other species of the  A. adloffispecies group: 9.8–18.8% (Appendix 2, Table 1S). Range of intraspecific distances: 0.2– 1.0% (Appendix 2, Table 2S).   Description.Morphometric and meristic data summarized in Table 2. Largest male examined 40.2 mmSL, largest female 39.7 mmSL. Dorsal profile slightly convex from snout to end of dorsal-fin base, straight on caudal peduncle. Ventral profile convex from lower jaw to origin of anal fin base, nearly straight on caudal peduncle. Greatest body depth at anal fin origin in males, at dorsal fin origin in females. Body deep and compressed. Eye positioned on lateral portion of head. Snout blunt and jaws short. Distal portion of dorsal fin rounded in both sexes. Dorsal-fin rays in males 18–22; in females 16–19. Dorsal-fin origin at vertical through 2nd to 4th anal fin rays in males, at vertical of urogenital papilla in females. Origin of dorsal fin at vertical through neural spines of 8th to 10th vertebrae in males, through neural spines of 9th to 12th vertebrae in females. Anal-fin rays in males 21–25, in females 18–23. Anal-fin tip rounded in males. In females, anal fin sub-triangular with antero-median rays lengthened, distal portion thickened. Origin of anal fin at vertical through pleural ribs of 7th to 9th vertebrae in males, through pleural ribs of 8th-10th vertebrae in females. Caudal fin rounded, 19–23 rays in both sexes. Pectoral fin elliptical with 11–12 rays in males and females. Pectoral-fin posterior tip reaching from 2nd to 6th anal fin ray in males, from origin of urogenital papilla to 2nd anal fin ray in females. Pelvic fin rays 5–6. Pelvic-fin posterior tip from 2nd to 4th anal-fin ray in males, from urogenital papilla to 5th anal fin ray in females. Pelvic-fin medial membrane ranging from slightly (about 10%) to almost completely (about 80%) coalesced. Urogenital papilla cylindrical and short in males, and pocket-shaped in females. Scales cycloid. Body and head entirely scaled, except for anteroventral surface of head. No scales on dorsal fin base, one irregular row of scales at anal fin base, and 2–3 rows of scales at caudal-fin base. Frontal squamation F, G or H patterned, sometimes irregularly arranged; E-scales slightly overlapping medially. One or two supra-orbital scales. Lateral line of trunk complete, with one neuromast per scale. Longitudinal series of scales 25–29; transverse series of scales 11–13; scale rows around caudal peduncle 14–16. Contact organs throughout antero-ventral part of flank in males. Row of minute contact organs in the 2–4 uppermost pectoral-fin rays in males. No contact organ on pelvic, dorsal and caudal fin. Cephalic neuromasts: supraorbital 15–21, parietal 1–4, anterior rostral 1, posterior rostral 1, infraorbital 2–3+24– 31, preorbital 2–3, otic 2–3, postotic 2–5, supratemporal 1, median opercular 1, ventral opercular 2–4, preopercular 20–28, mandibular 10–15, lateral mandibular 3–9, paramandibular 1. Two neuromasts on caudal-fin base. Six branchiostegal rays. Dermosphenotic ossification absent. Vomerine teeth absent. Urohyal deep. Total number of vertebrae 27–28, precaudal vertebrae 12–13. Gill rakers in first branchial arch 3 + 8–10. Basihyal subtriangular, width about 55–65% of length; basihyal cartilage about 60–70% of total basihyal length. One to three teeth on second pharyngobranchial.    TABLE II. Morphometric and meristic data of the holotype and paratypes of  Austrolebias lourenciano.    Morphometric data Holotype Males (n=11) Females (n=12)  Standart length (mm) 37.0 25.2–40.2 27.3–39.7  Percents of standart length  Body depth 37.3 34.8–40.8 33.4–41.9  Caudal peduncle depth 13.6 12.8–15.0 11.4–14.2  Predorsal length 53.7 53.3–56.9 58.4–63.4  Prepelvic length 43.6 42.3–47.4 45.2–53.2  Dorsal fin base length 36.8 35.4–41.8 26.9–29.4  Anal fin base length 39.1 38.1–43.8 25.8–29.9  Caudal fin length 30.2 30.5–36.2 31.4–38.1  Pectoral fin length 23.1 23.1–28.1 24.3–30.5  Pelvic fin length 9.6 8.9–11.6 10.8–15.4  Head length 29.3 28.7–32.3 28.5–32.4  Percents of head length  Head depth 105.3 97.1–106.6 95.7–105.3  Head width 58.7 53.1–65.2 58.6–71.3  Snout length 21.7 15.2–22.9 17.2–24.3  Lower jaw length 21.0 15.5–24.0 17.5–23.8  Eye diameter 28.4 28.3–31.7 27.3–32.7  Coloration in life.Males ( Figs. 5and 6): Ground colour of body light gray with lilac hues, mainly in the posterior region, and presence of 7–10 dark gray to black transversal bars. Pattern of transversal bars variable, ranging from presenting only well-defined bars (e.g., Fig. 5a), to also presenting narrower bars between well-defined bars ( Fig. 5b). Some specimens exhibit an inverse ground colour pattern, with a darker background (dark gray) and vertical light bars mainly in the posterior region of body ( Fig. 6b). Urogenital papilla gray. Pectoral and ventral region whitish. Opercular region greenish blue. Iris orange yellow, with black vertical band crossing eye. Dorsal fin gray or yellowish green with light yellow or light bluish bars forming small triangles, interspersed with small dark brownish row in dorsal fin base; presence of a black blotch on the distal portion of last dorsal fin rays bordered anteriorly by a narrow light blue margin. Anal fin gray or bluish gray with some iridescence, limited to basal region of anal fin, sometimes as an extension of the transversal bars of the body; sometimes iridescence absent, with only a single light yellow or gray basal band on anal fin. Presence of a black blotch on distal portion of last anal fin rays, bordered anteriorly by a narrow light blue margin. Caudal fin greenish blue or bluish gray, without dots, spots, bands or any marks; distal region hyaline. Pelvic fins greenish blue. Pectoral fins hyaline, with black margin.   FIGURE 5.Colour variation of males topotypes of  A. lourenciano(not preserved) captured in temporary pool in the floodplain of Arroio Pinto, southern Brazil. Photos by Matheus Volcan.   FIGURE 6.Largest paratype male (40.2 mm SL, MCP 54439) (A), and male with inverse ground color pattern in the posterior region of body (B) of  A. lourenciano, São Lourençodo Sul, Rio Grande do Sul, Brazil. Photo by Matheus Volcan. Females ( Fig. 7): Ground colour of body light brownish gray with dark markings, highly variable in size, number and shape. Sometimes two black spots on anterocentral portion of flank. Ventral region whitish. Opercular region ochre or pale greenish blue. Caudal peduncle with two vertically arranged black spots (sometimes coalesced into a eight-shaped spot) on posterior portion, absent in some specimens. Opercular region pale blue. Iris light yellow, with dark gray vertical band crossing the eye. Unpaired fins hyaline, with faint gray marks or bars, mainly on basal portion of dorsal and anal fins. Pectoral fins hyaline.   FIGURE 7.Variation in color pattern of females topotypes (not preserved) of  A. lourencianocollected in the floodplain of Arroio Pinto, Laguna dos Patos basin, Rio Grande do Sul, Brazil. Photo by Matheus Volcan.  Geographic distribution.  Austrolebias lourencianois only known from temporary wetlands of Arroio Pinto, Arroio São Lourenço basin, Laguna dos Patos system, Rio Grande do Sul, Brazil( Fig. 3)  Habitat notes.  Austrolebias lourencianooccurs in temporary wetlands with dense aquatic vegetation directly exposed to sunlight ( Fig. 8). Temporary wetlands inhabited by  A. lourencianousually dry in late spring and early summer, between November and December and flood again in early autumn between April and May, however, there is some variation from year to year depending on the intensity of the annual precipitations. The pools range from 20 to 50 cmin depth and have a muddy substrate. The areas from where the species was recorded are located at the edge of the riparian forest of the Arroio Pinto. The species was recorded co-occurring with another species of Rivulidae,  Austrolebiasaff. gymnoventris, in samples taken in 2014, although the last species was no longer recorded in the area after a portion of the typelocality was drained. Non-annual fish species recorded syntopically with the new species were  Corydoras paleatus(Jenyns, 1842),  Phalloceros caudimaculatus(Hensel, 1868),  Jenynsia lineata(Jenyns, 1842)and  Cheirodon ibicuhiensisEigenmann, 1915.   Etymology.The species name is derived from Portuguese adjective “lourenciano”, which is as the inhabitants from São Lourenço do Sul, a municipality in the state of Rio Grande do Sul, situated on the shores of the Laguna dos Patos, are known. The name  lourencianois a reference to the occurrence of the new species in this municipality. A name in apposition.  Conservation status.The species was relatively abundant in its typelocality. However, in 2015 a portion of the wetland was drained ( Fig. 8), reducing the flooded area. After the partial drainage of the temporary pool, a visible reduction in the density of the species was observed in subsequent years. In the second location where the species was recorded, on the opposite bank of the Arroio Pinto, there are small, shallow pools scattered in a pastureland, where few specimens are usually found. Both locations are subject to grazing and trampling by cattle. The species currently has an occupation area of less than 1 ha, with a low population density and suffers from the reduction in the quality of its habitat. Moreover, if measures to conserve the area of occurrence of  A. lourencianoare not taken, it is possible that the owner will carry out new interventions that will further reduce the distribution of the species. Thus, following the IUCN (2019),  A. lourencianopresents a reduced (AOO < 10,000 m²) and severely fragmented (a) area of occurrence and is experiencing continued decline (b) in the area of occupancy (ii) and in the quality of habitat (iii).  Austrolebias lourencianoshould be considered a “Critically Endangered” species under the category CR B2ab (ii, iii), and as such, the species should be included in future lists of the endangered fauna from Brazil. 3107836308 2013-09-30 V M. V. Volcan & A. C. Goncalves Brazil -31.347223 Arroio Pinto 20 -52.06472 Sao Lourenco do Sul 8 95 MCP 5440 1 Rio Grande do Sul holotype 3107836309 [199,1433,1782,1808] Sao Lourenco do Sul & Laguna dos Patos Brazil All 8 95 1 Rio Grande do Sul paratype 3107836306 MCP 54441 [151,1432,1818,1845] 2013-09-30 MCP M. V. Volcan & A. C. Goncalves Brazil -31.347223 Arroio Pinto 20 -52.06472 Sao Lourenco do Sul 8 95 11 6 5 Rio Grande do Sul paratype 3107836302 MCP 54442 [151,1287,1854,1881] 2013-09-30 MCP M. V. Volcan & A. C. Goncalves Brazil -31.347223 Arroio Pinto 20 -52.06472 Sao Lourenco do Sul 8 95 12 6 6 Rio Grande do Sul paratype 3107836307 2013-09-03 V A. C. Goncalves Brazil Volcan 8 95 1 Rio Grande do Sul paratype 3107836312 MCP 54439 2013-09-03 MCP A. C. Goncalves Brazil Volcan 8 95 2 1 1 Rio Grande do Sul paratype 3107836304 [273,616,1926,1952] V Volcan, G. M & Wallwitz Brazil 8 95 1 Rio Grande do Sul paratype 3107836310 [626,899,1926,1952] 2018-08-16 T Brazil Basi 8 95 1 Rio Grande do Sul paratype 3107836301 MCP 54443 MCP Arroio Pinto Brazil -31.346668 20 -52.06333 8 95 4 3 1 Rio Grande do Sul paratype 3107836305 [336,881,1998,2026] 2013-09-03 V A. C. Goncalves Brazil Volcan 8 95 1 Rio Grande do Sul paratype