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 [151,394,223,250] Actinopterygii Rivulidae Austrolebias Animalia Cyprinodontiformes 3 90 Chordata species cheffei sp. nov.     Holotype. MCP 54437, male, 30.8 mmSL, Brazil, Rio Grande do Sul State, Cristalmunicipality, temporary pool in the floodplains of Rio Camaquã, Laguna dos Patos system, 31°01’48”S 52°00’32”W, L.E.K. Lanés& B. Klotzel,  28 October 2015.    Paratypes. MCP 54438, 6 males 24.7–33.1 mmSL (1 C&S), 7 females(1 C&S), 26.4–33.1 mmSL, same data as holotype.   Diagnosis.The new species differs from all other species of the genus except  A. adloffi,  A. charrua,  A. lourenciano, 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. black blotches absent in the remaining species).  Austrolebias cheffeiis distinguished from the remaining species of the  A. adloffispecies group by presenting a yellowish green or yellowish blue dorsal fin with wide black bars extending from the base to the median region of the fins in males (vs. never a similar pattern); and, except from  A. reicherti,by presenting anal fin blue with 3–6 incomplete, wide black transversal bars (each bar covering 3–6 rays), limited to basal and medial anal fin regions, which consist of transversal flank bars extensions (vs. never a similar pattern); from the other species of the  A. adloffispecies group, except  A. nachtigalli, A. nigrofasciatus, A. pongondo, A. lourenciano, and  A. reicherti, by the presence of a black blotch on the distal portion of the last anal and dorsal fin rays bordered anteriorly by a small light blue margin (vs. black blotch absent or inconspicuous in the last anal and dorsal fin rays).  Austrolebias cheffeiis also distinguished from all other species of the  A. adloffispecies group, except  A. pelotapes,by having the urogenital papilla attached to the anal fin in males ( vs. urogenital papilla free), and except from  A. nigrofasciatusand  A. reichertiby dorsal-fin origin anterior to anal-fin origin in males (vs. dorsal-fin origin posterior to anal-fin origin). In addition, the following characters are useful for distinguishing  A. cheffeifrom the other species of the  A. adloffispecies group: from  A. adloffiby separated preopercular and mandibular series of neuromasts (vs. united mandibular and preopercular series), and by higher head length in females (31.1–33.5% SL, vs. 28.6–30.5% SL). Autrolebias cheffeidiffers from  A. charruaby separated preopercular and mandibular series of neuromasts (united vs. mandibular and preopercular series), and by higher head length (31.1–33.5% SL, vs. 26.5– 30.5 % SL), and lower caudal fin rays in females (17–20 vs. 23–26). Autrolebias cheffeidiffers from  A. lourencianoby the greater number of rays in the anal fin of males (25–27 vs. 21–24). It differs from  A. minuanoby separated preopercular and mandibular series of neuromasts (vs. united mandibular and preopercular series), by higher head length (30.5–33.5 % SL, vs. 26.0–30.4 % SL) and lower head depth in males (100.0–109.3 % HL, vs 112.3–138.7 % HL), and by higher head length (31.1–33.5 % SL, vs. 27.7–30.1 % SL) and less caudal fin rays in females (17–20 vs. 21–25). Autrolebias cheffeidiffers from  A. nachtigalliby the absence of golden to pink iridescence at the distal half of dorsal fin in males (vs. presence of golden to pink iridescence in distal half of dorsal fin), by presenting a dark bluish-gray caudal fin, without dots or spots in males (vs. light blue iridescence and faint, elongated blue spots), by a higher head length (30.5–33.5% SL, vs. 26.1–29.0% SL), lower head depth (100.0–109.3% HL, vs. 113.7–132.1% HL), a lower head width in males (58.4–65.8% HL, vs. 66.6–71.4% HL), higher head length (31.1–33.5% SL, vs. 27.8–30.5% SL), and less caudal fin rays in females (17–20 vs. 21–24). It differs from  A. nigrofasciatusby a lower head depth in males (100.0–109.3% HL, vs. 110.6–121.6 % HL), by the absence of blue iridescence on the distal half of anal fin (vs. presence of blue iridescence on the distal half of anal fin), by presenting a greenish blue caudal fin, without dots or spots in males (vs. elongated light blue spots), and by higher head length in females (31.1–33.5% SL, vs. 27.8–30.8 % SL). It differs from  A. reichertiby higher dorsal fin base length (36.0–42.6% SL, vs. 34.2–34.9 % SL) in males and by higher body depth (36.8–42.3% SL, vs. 29.5–36.1 % SL) in females. Autrolebias cheffeidiffers from  A. pelotapesby separated preopercular and mandibular series of neuromasts (vs. united mandibular and preopercular series), and by dorsal fin origin at vertical between pelvic fin origin and anus in females (vs. between base of 1st and 2nd rays of anal fin). Autrolebias cheffeidiffers from  A. pongondoby a lower head depth in males (100.0–109.3% HL, vs. 111.0–122.0% HL), higher head length (31.1–33.5% SL, vs. 27.5–30.8 % SL), lower head width (62.3–71.5% HL, vs. 73.0–81.0% HL), less caudal fin rays (17–20 vs. 22–23), and by dorsal fin origin at vertical between pelvic fin origin and anus in females (vs. between base of 1st and 3rd rays of anal fin).   Austrolebias cheffeiis distinguished from all other species or lineages of the  A. adloffispecies group by one unique nucleotide substitution along the cytb alignment: cytb.309 (T> C) (Supporting file, Fig. S2). It is distinguished from  A.aff. minuano1 by this and two other nucleotide substitutions: cytb.319 (T> C) and cytb.406 (G> A). In addition, it is distinguished from  A. lourencianoby these three in addition to other six substitutions [cytb.133 (C> A), cytb.304 (C> T), cytb.319 (T> C), cytb.406 (G> A), cytb.457 (G> T), cytb.466 (A> G), cytb.586 (A> G) and cytb.595 (T> C)].  Genetic distance.From  A.aff. minuano1: 1.5–2.6%; from  A. lourenciano: 1.9–2.6%; from  A.aff. minuano2: 2.9–3.8%; from  A. adloffi sensu lato:3.1–3.4%; from  A. pongondo: 3.6–4.7 %; from  A. pelotapes:4.5–4.9 %; from  A. bagual: 6.2–6.8%; from  A. nigrofasciatus:7.5–9.4%; from  A. minuano sensustricto: 10.4–11.2%; from other species of the  A. adloffispecies group: 10.6–20.5% (Appendix 2, Table 1S). Range of intraspecific distances: 0.2–0.7% (Appendix 2, Table 2S).   Description.Morphometric and meristic data summarized in Table 1. Largest male examined 33.1 mmSL, largest female 33.1 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 dorsal fin origin in males, at pelvic fin base 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 22–24; in females 18–21. Dorsal-fin origin at vertical of urogenital papilla in males, at vertical of pelvic fin base or anus in females. Origin of dorsal fin at vertical through neural spines of 6th and 7th vertebrae in males, in females through neural spines of 9th and 10th vertebrae. Anal-fin rays in males 25–27, in females 21–24. Anal-fin tip rounded in males. In females, anal fin subtriangular with antero-median rays lengthened, distal portion thickened. Origin of anal fin at vertical through pleural ribs of 6th and 7th vertebrae in males, in females through pleural ribs of 9th-10th vertebrae. Caudal fin rounded, 19–22 rays in males, 17–20 infemales. Pectoral fin elliptical with 11–12 rays in males, 10–13 infemales. Pectoralfin posterior tip reaching from 4th to 6th anal fin ray in males, from origin of urogenital papilla to 4th anal fin ray in females. Pelvic fin rays 5. Pelvic-fin posterior tip from 3rd to 4th anal-fin ray in males, from 2nd to 5th anal fin ray in females. Pelvic-fin medial membrane about 10–50% coalesced. Urogenital papilla cylindrical and short in males; urogenital papilla pocket-shaped in females. Urogenital papilla attached from base to 30% of its length to anal fin in males.   TABLE I. Morphometric and meristic data of the holotype and paratypes of  Austrolebias cheffei.    Holotype Males (n=6) Females (n=7)  Morphometric data  Standart length (mm) 30.8 24.4 –33.1 26.4 –33.1  Percents of standart length  Body depth 38.8 38.8–42.0 36.8–42.3  Caudal peduncle depth 13.3 13.3–15.8 12.5–14.8  Predorsal length 50.0 49.9–55.9 57.0–63.4  Prepelvic length 45.3 45.3–47.7 50.5–55.9  Dorsal fin base length 42.6 36.0–42.6 27.0–32.9  Anal fin base length 45.1 39.4–45.1 28.5–32.9  Caudal fin length 30.1 28.8–35.1 33.3–39.3  Pectoral fin length 26.6 23.4–28.5 24.6–29.1  Pelvic fin length 9.9 8.8–11.6 12.5–15.2  Head length 30.8 30.5–33.5 31.1–33.5  Percents of head length  Head depth 105.4 100.0–109.3 95.8–104.5  Head width 60.3 58.6–65.8 62.3–71.5  Snout length 19.4 18.6–22.5 17.1–21.6  Lower jaw length 19.1 19.1–22.9 16.5–21.8  Eye diameter 32.7 29.6–34.0 28.6–30.3   FIGURE 1.Colour variation of male topotypes (not preserved) of  Austrolebias cheffei, floodplain of Rio Camaquã basin, Cristal, Rio Grande do Sul, Brazil. Photos by Pedro Hoffmman (A) and Matheus Volcan (B–D).   FIGURE 2.Females topotypes (not preserved) of  Austrolebias cheffei, floodplain of Rio Camaquã basin, Cristal, Rio Grande do Sul, Brazil. Photo by Matheus Volcan. Scales cycloid. Body and head entirely scaled, except anteroventral surface of head. No scales on dorsal fin base, one irregular row of scales in anal fin base in males, in females scales absent in anal fin base, and 3 rows of scales on caudal-fin base. Frontal squamation F or G, sometimes irregularly arranged; E-scales slightly overlapping medially. Two supra-orbital scale, except one individual with one supra-orbital scale. Lateral line of trunk complete, with one neuromast per scale. Longitudinal series of scales 25–30; transverse series of scales 12–13; scale rows around caudal peduncle 14–16. Contact organs throughout antero-ventral part of flank in males. Rows 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 16–22, parietal 2–4, anterior rostral 1, posterior rostral 1, infraorbital 2–3+20– 30, preorbital 2–3, otic 2–4, postotic 1–5, supratemporal 1, median opercular 1, ventral opercular 1–3, preopercular 18–26, mandibular 10–15, lateral mandibular 4–6, paramandibular 1. Two neuromasts on caudal-fin base. Six branchiostegal rays. Dermosphenotic ossification absent. Vomerine teeth absent. Urohyal deep. Total number of vertebrae 28. Gill rakers in first branchial arch 3 + 8–9. Basihyal subtriangular, width about 55–60% of length; basihyal cartilage about 45–50% of total basihyal length. Two or three teeth on second pharyngobranchial.  Coloration in life.Males ( Fig. 1): Ground colour of body light gray, yellowish in some specimens, with 5–9 dark gray to black transversal bars, that extend into the basis of dorsal and anal fins as blurred bars. Dorsum brownish gray. Urogenital papilla gray. Pectoral and ventral region whitish or yellowish. Opercular region blue. Iris brownish orange. Black vertical band crossing the eye. Supraorbital and suborbital bars black. Dorsal fin blue, with yellowish iridescent pigmentation and 3–6 wide, dark-gray to black vertical bars (each bar covers between 3–7 rays), which consist of transverse flank bars extensions, usually restricted to basal region, and sometimes reaching the middle region. Vertically elongated dark spot at distal portion of last dorsal fin rays, anteriorly delimited by a narrow whitish blue margin. Anal fin bluish with the same bars pattern as the dorsal fin. Presence of vertically elongated dark spot at distal portion of last anal fin rays, anteriorly delimited by a narrow whitish blue margin. Caudal fin blue, sometimes with horizontally whitish iridescent stripes along caudal fin rays; distal region hyaline. Pelvic fins blue. Pectoral fins hyaline with black margin. Females ( Fig. 2): Ground colour of body light brownish gray with dark marks, highly variable in size, number and shape. Sometimes one or two black spots on anterocentral portion of flank. Pectoral and ventral region hyaline. Opercular region ochre or pale greenish blue. Caudal peduncle with two vertically arranged black spots (sometimes coalescing into a eight-shaped spot) on posterior portion. Opercular region pale blue. Iris light yellow. 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.   Geographic distribution.  Austrolebias cheffeiis endemic of temporary wetlands of the floodplains of lower course of the Rio Camaquã basin, Laguna dos Patos system, Rio Grande do Sul, Brazil( Fig. 3).   Habitat notes.  Austrolebias cheffeiwas recorded in wetlands in the lower portion of the Rio Camaquã ( Fig. 4). The species inhabits small (less than 1 ha), shallow temporary wetlands (< 60 cmdeep), with high density of aquatic vegetation, exposed to sunlight, on the edge of the riparian forest of the Rio Camaquã, but it is also found in small pools within the riparian forest. Temporary wetlands with  A. cheffeiusually 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 species was recorded co-occurring with another two species of Rivulidae,  Austrolebiasaff. wolterstorffi(Ahl, 1924)and  Cynopoecilus melanotaenia(Regan, 1912).   Etymology.The specific epithet is given in honour of Morevy Moreira Cheffe for his contribution to the knowledge of annual fish in southern Brazil, especially the  A. adloffispecies group. To him our gratitude and our acknowledgement.   Conservation status.The species was found in small wetlands in a rural landscape matrix highly degraded by rice and soybean cultivation. The species occurs in lowland areas, which are widely used for agriculture and their populations are considered relictual. Several collection expeditions were carried out in the lower Rio Camaquã basin and  A. cheffeiwas only recorded at its typelocality. The very few wetlands still remaining in the region of the lower Rio Camaquã is probably due to the massive loss of this typeof environment that was drained for irrigated rice crops, and more recently for soybean crops. It is probable that the species occurs in the Parque Estadual do Camaquã, about 25 kmdownstream from the typelocality of  A. cheffei, and collection efforts in the area are necessary to ascertain its occurrence in this conservation unit. The species has an occupation area of less than 1 ha, a low population density and suffers from the reduction in the quality of its habitat. Following the IUCN (2019),  A. cheffeipresents 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), and should be considered as a “Critically Endangered” species under the category CR B2ab (ii, iii). The species should be included in future lists of the endangered fauna from Rio Grande do Suland Brazil. 3107836303 2015-10-28 MCP L. E. K. Lanes & B. Klotzel Brazil -31.03 Rio Camaqua 20 -52.00889 Cristal 3 90 MCP 54437 1 Rio Grande do Sul State holotype 3107836311 2015-10-28 MCP L. E. K. Lanes & B. Klotzel Brazil -31.03 Rio Camaqua 20 -52.00889 Cristal 3 90 MCP 54438 13 7 6 Rio Grande do Sul paratype