From the mud to the tree: phylogeny of Austrolebias killifishes, new generic structure and description of a new species (Cyprinodontiformes: Rivulidae) Author Alonso, Felipe Instituto de Bio y Geociencias del NOA (IBIGEO), National Scientific and Technical Research Council (CONICET) - UNSa, Av. 9 de Julio 14, A 4405 BBA Rosario de Lerma, Salta, Argentina & Killifish Foundation, La Plata, Buenos Aires Province, Argentina Author Terán, Guillermo Enrique Killifish Foundation, La Plata, Buenos Aires Province, Argentina & Fundación Miguel Lillo - Unidad Ejecutora Lillo (CONICET), San Miguel de Tucumán, Tucumán, Tucuman Province, Argentina Author Alanís, Wilson Sebastián Serra Killifish Foundation, La Plata, Buenos Aires Province, Argentina & Sección Ictiología, Departamento de Zoología, Museo Nacional de Historia Natural, Montevideo, Uruguay & Centro Universitario Regional del Este (CURE) Sede Rocha, Rocha, Uruguay Author Calviño, Pablo Killifish Foundation, La Plata, Buenos Aires Province, Argentina Author Montes, Martin Miguel Killifish Foundation, La Plata, Buenos Aires Province, Argentina & Centro de Estudios Parasitológicos y de Vectores - [CCT-CONICET - La Plata] Centro Científico Tecnológico Conicet, La Plata - [Conicet] Consejo Nacional De Investigaciones Científicas y Técnicas, La Plata, Buenos Aires Province, Argentina Author García, Ignacio Daniel Killifish Foundation, La Plata, Buenos Aires Province, Argentina & Instituto de Limnología ‘ Dr. Raúl Ringuelet’ (ILPLA) - UNLP-CONICET, La Plata, Buenos Aires Province, Argentina Author Barneche, Jorge Adrián Killifish Foundation, La Plata, Buenos Aires Province, Argentina & Centro de Estudios Parasitológicos y de Vectores - [CCT-CONICET - La Plata] Centro Científico Tecnológico Conicet, La Plata - [Conicet] Consejo Nacional De Investigaciones Científicas y Técnicas, La Plata, Buenos Aires Province, Argentina Author Almirón, Adriana Universidad Nacional de La Plata, Facultad de Ciencias Naturales y Museo, División Zoología Vertebrados, La Plata, Buenos Aires Province, Argentina Author Ciotek, Liliana Administración de Parques Nacionales, Parque Nacional Campos del Tuyú (7103), General Lavalle, Buenos Aires Province, Argentina Author Giorgis, Pablo Administración de Parques Nacionales, Parque Nacional Campos del Tuyú (7103), General Lavalle, Buenos Aires Province, Argentina Author Cascioưa, Jorge Universidad Nacional de La Plata, Facultad de Ciencias Naturales y Museo, División Zoología Vertebrados, La Plata, Buenos Aires Province, Argentina text Zoological Journal of the Linnean Society 2023 2023-07-10 199 1 280 309 http://dx.doi.org/10.1093/zoolinnean/zlad032 journal article 266147 10.1093/zoolinnean/zlad032 684543f4-855c-492a-b163-a157473a187c 0024-4082 8326346 E1210342-9DBD-4D89-ABF5-1925792D0E14C Argolebias guarani Alonso, Calviño, Terán, Serra, Montes, García, Barneche, Almirón, Ciotek, Giorgis & Casciofla sp. nov. ( Figs 1 , 3–11 ; Tables 2–4 ) LSID urn:lsid:zoobank.org:act: 1F9F0132-D626-40D7-8511- 14123321C8E1 Holotype : IBIGEO-I 473 , 51.0 mm SL, male; Argentina : Misiones Province : Reserva Natural de la Defensa Puerto Península : seasonal pond on the side of an internal road ( −25.677114 , −54.578445 ), Middle Paraná River basin. Elevation : 179 m a.s.l. ; Coll. : F. Alonso , M. Montes, J . Casciotta, A . Almirón, P . Giorgis, L. Ciotek. October 2018 . Paratypes : IBIGEO-I 474, one male 48.0 mmSL, eight females 32.8–35.3 mm SL; MLP11436 , one male 55.2 mm SL, three females 33.8–35.2 mm SL; CI-FML 7805, eight males , 29.9– 48.6 mm SL, three females 26.5–27.8 mm SL; one male (c&s), 44.7 mm SL, one female (c&s) 37.6 mm SL; all collected with the holotype . MACN-Ict 12712, one male , 47.4 mm SL, four females 30.2–37.3 mm SL, same locality as the holotype, Coll. : J. Barneche , M. Montes, P . Marcotegui, 23 March 2018 . IBIGEO-I 475, seven males 26.9–33.7 mm SL, one female 30.7 mm SL, 18 November 2017 , Coll : J. Casciotta , A. Almirón , M. Montes , P. Giorgis , L. Ciotek . Diagnosis Argolebias guarani can be distinguished from all other congeners by presenting a unique colour pattern ( Figs 4–6 ) consisting of the following: in mature alive males, (1) scattered small dark brown to grey irregular blotches on the laterodorsal surface of head and anterior portion of trunk; (2) abundant and relatively big sub-square irregular iridescent turquoise to light blue blotches on unpaired fins, evenly distributed, with those in the distal portion elongated and merging; (3) trunk and tail with anterocentral portion of scales iridescent turquoise with grey borders generating a reticulated pattern; (4) thin external border of scales in dorsal portion of trunk, below dorsal fin, iridescent turquoise; and by (5) ventrolaterally directed anterior nostrils; and, in juveniles and young adults, (6) orange-yellowish background coloration on the base of unpaired fins and yellowish belly. Further comparisons with species of Argolebias Argolebias guarani is similar and, based on our phylogenetic analysis, closely related to Argolebias nigripinnis and Argolebias paranaensis , sharing with Argolebias nigripinnis a unique character of preopercular and mandibular series of neuromasts separated (not evaluated in Argolebias paranaensis ). This new species can be differentiated from Argolebias nigripinnis further by not presenting a bright iridescent subdistal stripe on the dorsal fin (vs. present) and by presenting an infraorbital bar narrower than the width of the orbit (vs. its dorsal portion approximately as wide as orbital diameter). It can be differentiated from Argolebias paranaensis by presenting the dorsal fin origin anterior to the anal fin origin (vs. posterior), more pectoral fin rays (12–13 vs. 10–11), shorter caudal fin length (22.9–28.1 vs. 34.5–35.3 in males; and 22.8–30.6 vs. 31.9–34.7 in females, in percentages of SL) and smaller eye diameter (27.2–31.6 vs. 34.8–38.4 in males; and 26.6–31.4 vs. 37.2–38.0 in females in percentages of HL). Also, Argolebias guarani can be differentiated further from Argolebias nigripinnis by the following: (1) presenting more pectoral fin rays (12–13 vs. 10–11); (2) a shorter caudal fin length (22.9–28.1 vs. 31.9–34.7 in males, and 22.8–30.6 vs. 31.9–34.7 in females, in percentages of SL); (3) a smaller eye diameter (27.2–31.6 vs. 32.8–37.1 in males, and 26.6–31.4 vs. 33.0– 36.9 in females, in percentages of HL); (4) the presence of vertically elongated, dark grey blotches on the posterocentral portion of the trunk in females (vs. absent); (5) trunk and tail of mature males with anterocentral portion of scales iridescent turquoise with grey borders, generating a reticulated pattern (vs. black to grey general background with some scales with light blue anterocentral portion with grey borders arranged in irregular vertical lines); (6) a different colour pattern on the dorsal fin consisting of abundant, evenly distributed, relatively big and sub-square irregular iridescent turquoise to light blue blotches, with those in the distal portion elongated and merging (vs. fewer, smaller and rounded light blue blotches, mainly concentrated on the basal portion, and a subdistal light blue band); (7) dorsal portion between preopercle and opercle iridescent turquoise (vs. brownish); and (8) infraorbital bar thin and triangular (vs. wider, approximately same as eye width, and diffuse). Description Morphometric data are provided in Table 2 . Morphometric and meristic data are given in the Supporting Information (Appendix S9). Males are larger than females (largest examined male 64.8 mm SL, largest female 48.3 mm SL). Body elongate, moderately deep, laterally compressed. Highest body depth at dorsal fin origin. Dorsal profile on lateral view, concave from snout to vertical through anterior margin of operculum, convex from this point to posterior end of dorsal fin base, and approximately straight on caudal peduncle. Ventral profile on lateral view, convex from lower jaw to end of anal fin base, nearly straight on anal fin base, and straight on caudal peduncle. Snout blunt and jaws short. Dorsal fin length in anterior portion shorter than in posterior portion. Posterior distal border of dorsal fin generally slightly pointed in males. Dorsal fin serrated. Anal fin subrectangular, with rounded anterior distal border. Posterior distal border of anal fin pointed in both sexes. Anteromedial rays of anal fin of females not lengthened. Anal fin distal border serrated in males and slightly serrated in females. Caudal fin relatively short, subtriangular and rounded. Pelvic fin bases close and not united. Anal fin origin posterior to dorsal fin origin in males, on vertical between base of fourth to seventh dorsal fin rays; dorsal fin origin anterior to anal fin origin in females on vertical between base of second to sixth anal fin rays; dorsal fin origin at vertical between fifth and sixth pleural ribs and ninth and 10th vertebrae in males and between ninth and 10th pleural ribs and ninth and 10th vertebrae in females. Base of anal fin origin at vertical between seventh and eighth pleural ribs in males and between 12th and 13th pleural ribs in females. Urogenital papillae not attached to anal fin, sometimes reaching base of first anal fin ray. Pectoral fins elliptical, posterior margin on vertical between urogenital papilla and base of fourth anal fin ray in males, reaching or almost reaching pelvic fin base in females. Tip of each pelvic fin reaching base of first or second anal fin rays in males, between urogenital papillae and base of first anal fin ray in females. Between 24 and 26 dorsal fin rays in males and between 17 and 20 in females. Between 22 and 25 anal fin rays in males and 19 or 20 in females. Between 23 and 27 caudal fin rays (only two specimens with 27 and one with 23, N = 24). Between 12 and 14 pectoral fin rays (only one specimen with 14). Six pelvic fin rays. Figure 4. Argolebias guarani IBIGEO-I 473, 51.0 mm standard length, male, holotype, in left lateral view. A, picture of live animal taken in aquarium. B, holotype preserved in alcohol after fixation in formaldehyde. Figure 5. Argolebias guarani detail of diagnostic characters. A, orange-yellowish background coloration on the base of unpaired fins and yellowish belly in juveniles and young adults. B, scattered small dark brown to grey irregular blotches on the laterodorsal surface of the head and anterior portion of the trunk. C, abundant and relatively big sub-square irregular iridescent turquoise to light blue blotches on unpaired fins, evenly distributed, with those on the distal portion elongated and merging; trunk and tail of mature males with anterocentral portion of scales iridescent turquoise with grey borders, generating a reticulated pattern; and thin border of scales below dorsal fin in dorsal portion of trunk iridescent turquoise. Figure 6. Colour pattern variability of Argolebias guarani males. A, male immediately after capture. B–D, in aquarium. Figure 7. Colour pattern variability of Argolebias guarani females. A, immediately after capture. B–D, in aquarium. Scales large and cycloid. Trunk and head scaled, except for ventral surface of head. No scales on dorsal and anal fin bases, and two rows of scales on caudal fin base. Frontal squamation H-patterned, sometimes F; E-scales overlapping medially; scales arranged in transverse pattern. Longitudinal series of 23–27 scales, arranged regularly; transversal series of 10–12 scales; 15 or 16 rows of scales around caudal peduncle. One contact organ on distal posterior central portion of flanks scales in males, more prominent on ventral region. A row of minute contact organs on uppermost and second pectoral fin rays in males, sometimes absent; no contact organs on unpaired fins. Figure 9. Ontogenetic changes in colour patterns in Argolebias guarani females. A, juvenile ~1 cm standard length (SL). B, young adult ~1.5 cm SL. C, adult individual ~2.5 cm SL. Figure 8. Ontogenetic changes in colour patterns in Argolebias guarani males. A, juvenile ~1 cm standard length (SL). B, juvenile ~1.5 cm SL. C, juvenile ~2 cm SL. D, young adult ~2.5 cm SL. E, adult individual ~5 cm SL. Cephalic neuromasts: 14 supraorbital, one parietal, one anterior rostral, one posterior rostral, 28 infraorbital, two preorbital, two otic, one to three post-otic, one supratemporal, one median opercular, two ventral opercular, 19 or 20 preopercular, 10 or 11 mandibular and six lateral mandibular. Basihyal subtriangular, width ~30–35% of length; basihyal cartilage short or moderate, ~35% of total basihyal length, without lateral projections. Dermosphenotic ossification absent. Ventral process of posttemporal short. Six branchiostegal rays. Two teeth on second pharyngobranchial. Three plus nine gill rakers on first branchial arch. Vertebrae: total 26, comprising 11 abdominal and 15 caudal. Coloration in life Males Adult males ( Figs 4–6 ) present a markedly reticulated pattern on the body, with intense turquoise coloration. Scales on trunk, tail and dorsal portion of the head with dark grey borders. Anterocentral portion of most scales iridescent light blue to turquoise, except for those on the dorsal portion of the head and trunk, anterior to the dorsal fin origin, which present a grey central portion. The portion of the head posterior to the infraorbital band and ventral to the dorsal portion of the gill opening are light blue to turquoise. Small scattered irregular dark grey to brown blotches are present on the dorsal portion of the head. There is a dark brown blotch around the neuromast supraorbital series, posterior rostral series and orbital area. There is a short infraorbital band approximately as wide as the pupil. The pupil is black, horizontally elongated. There is a vertical black stripe on the eye, approximately the same width as the pupil. The iris is silver grey, with orange around the pupil. The ventral portion of the head and anterior abdominal region are light blue to whitish. The area over the dentary, maxilla and premaxilla is grey. The fins have a well-defined black border. With abundant, evenly distributed, relatively big and sub-square irregular iridescent turquoise to light blue blotches. Those in the distal portion are elongated and fused. The base of unpaired fins is orange in young individuals; grey with an orange hue in mature males. The first dorsal fin ray is dark black. The distal portion of dorsal fin rays is dark black, more marked on anterior rays, which can be completely black in some specimens. The pectoral fin is light grey, translucent, with a light blue to turquoise hue and light blue blotches on the medial distal portion, which, in some specimens, are scattered along the whole fin. Pelvic fins are dark grey with irregular light blue to turquoise blotches. Figure 10. Males of Argolebias guarani after fixation, preserved in 70% ethanol. Females Females ( Fig. 7 ) present a yellow to orange background coloration of the body and the basal portion of unpaired fins, which present light brown rays and a mainly hyaline interradial membrane. Anal and dorsal fins have light grey to light brown blotches, mainly concentrated on the basal portion and generally over the rays. Paired fins are hyaline. The caudal fin is hyaline. There are faint scattered irregular brown blotches on the trunk and tail. There are small, vertically elongated, dark grey blotches mainly concentrated on the central portion of the trunk. Thre is a small faint grey infraorbital band, thinner than the iris, which is light orange with a grey dorsal portion. The pupil is black. The ventral portion of the head and anterior ventral region are whitish. The opercle is light green, with scattered melanophores covering it. There is a dark brown blotch around the neuromast supraorbital series, posterior rostral series and orbital area. The area over the dentary, maxilla and premaxilla is grey. Ontogenetic changes in colour paflern Males Juveniles ~ 1 cm SL ( Fig. 8A ) present a yellow body background coloration and basal portion of unpaired fins. Paired fins are hyaline with black borders. There are faint light grey vertical bands on the trunk and tail, wider above the anal fin, interspaced with thin clearer bands. The dorsal and anal fin present some light yellow faint blotches on the base. At ~ 1.5 cm SL ( Fig. 8B ), the background coloration of the body and base of unpaired fins changes to a light orange. Individuals present more defined and darker grey vertical bands on the flanks. Some scales in the light vertical band present a slightly iridescent central portion. Light blue blotches appear in the unpaired fins and the base of the pelvic fins, while the background of all these becomes grey distally. The pectoral fins remain hyaline with black borders. At ~ 2 cm SL ( Fig. 8C ), the background body colour and base of unpaired fins becomes more orange. Most scales on light vertical bands show a light blue central portion. The portion of the head posterior to the infraorbital band and ventral to the dorsal portion of the gill opening turns light blue. The first dorsal fin ray turns black. In young adult males, ~ 2.5 cm SL ( Fig. 8D ), the body background coloration turns to light grey with a yellow abdominal portion. Scales on the trunk, tail and dorsal portion of the head have dark grey borders. Some scattered irregular dark grey to brown blotches appear on the dorsal portion of the head. The centre of most scales turns light blue, except for those on the dorsal portions of the head and trunk, anterior to the dorsal fin origin, which are grey. Vertical bars are no longer observable. The base of unpaired fins remains orange. Light blue blotches and markings become more intense, tending to turquoise. The fins have a well-defined black border. The medial distal portion of the pectoral fin has light blue markings. Adult males ( Fig. 8E ) have a markedly grey reticulated pattern on the body and intense turquoise coloration. The base of unpaired fins is grey, with an orange hue. Head markings are more intense. The pectoral fin has a greater number of light blue blotches. Females Young females, ~ 1.5 cm SL ( Fig. 9A ), present a yellow body background coloration and basal portion of the unpaired fins. The anal and dorsal fins present light grey blotches mainly concentrated on the basal portion and generally over the rays. Paired fins are hyaline. There are small, vertically elongated dark grey blotches on the central portion of the body. Figure 11. Females of Argolebias guarani after fixation, preserved in 70% ethanol. At ~ 2 cm SL ( Fig. 9B ), the background coloration becomes light orange, and small faint scattered irregular brown blotches appear on the body. At ~ 2.5 cm SL ( Fig. 9C ), the background coloration becomes a more intense light orange, and blotches tend to become darker. Coloration of preserved specimens in alcohol The coloration of preserved specimens in alcohol is similar to that of live specimens, but without iridescent coloration and with a general pale amber background with light grey markings ( Figs 10 , 11 ). Briefly, males have light grey fins with hyaline dots and markings arranged in the same pattern as the light blue to turquoise iridescent markings in live specimens described above. Most scales present a wide grey border and a thin most external clear border, more marked and contrasting in the dorsal portion of the body. There are irregular grey markings on the dorsal portion of the head and anterior dorsal portion of the trunk. There is a faint suborbital bar below the eye. Females have hyaline fins with sub-square grey markings on the basal portion of unpaired fins in the membranous portion between rays, tending to form an intercalated pattern, more conspicuous and with bigger blotches on the anal fin. The body has irregular grey blotches and darker grey blotches more superficial on the skin, concentrated on the trunk portion immediately anterior to the dorsal fin origin at the height of the eye. Some individuals have fewer and smaller dark grey blotches on the the ventral portion of the caudal peduncle. The dorsal portion of the head and trunk, anterior to the end of the dorsal fin, is dark brown, with scales presenting a thin light brown border. There is a faint suborbital bar. Distribution Argolebias guarani is known only from its type locality, a seasonal pond on the side of an internal road at Reserva Natural de la Defensa Puerto Península ( −25.677114 , −54.578445 ), Middle Paraná River basin, at an elevation of 179 m a.s.l. , in Misiones province , north-east Argentina . It is the northern- and eastern-most record for the genus Argolebias ( Fig. 12 ). Ecology The type locality is a seasonal pond, mainly covered with grass, in the Paraná Forest at ~ 185 m a.s.l., in an open space in the surrounding jungle, with abundant palm trees. The maximum depth measured was ~ 80 cm , and the water was clear. No submerged aquatic vegetation was observed. The bottom presented a soft sediment, with abundant organic matter ( Fig. 13 ). Table 2. Morphometric data for Argolebias guarani : holotype and paratypes ( N = 24; range, mean and SD include the holotype).

Parameter	Holotype	Male ( N = 10)	Female ( N = 14)
Range	Mean	SD	Range	Mean	SD
Total length (mm)	64.8	38.3–64.8	48.2	10	38.2–48.3	43.4	3.1
Standard length (mm)	51.0	29.9–51.0	38.5	8	30.2–37.3	34.2	2.3
Measurements as a percentage of standard length
Body depth	44.5	32.9–44.5	36.9	3.2	30.2–37.6	35.5	2.0
Caudal peduncle depth	18.4	14.4–18.4	15.6	1.3	14.0–15.8	14.8	0.6
Predorsal length	48.0	43.7–48.0	45.7	1.4	54.0–61.4	58.4	2.0
Prepelvic length	50.0	40.3–50.0	45.0	2.8	49.4–56.7	52.6	2.0
Preanal length	56.3	45.6–56.3	51.3	3.3	60.6–69.1	63.1	2.2
Length of dorsal fin base	44.7	42.5–54.4	46.5	3.3	24.8–35.0	30.3	2.9
Length of anal fin base	40.4	38.0–47.5	41.0	2.8	21.1–27.7	25.0	1.8
Caudal fin length	27.1	22.9–28.1	25.1	1.6	22.8–30.6	27.0	2.0
Pectoral fin length	21.4	18.0–25.8	21.4	2.3	19.6–26.5	21.8	1.7
Pelvic fin length	10.4	7.3–10.4	8.5	1.1	7.4–13.7	10.5	1.6
Head length	28.4	28.0–31.9	29.6	1.4	27.7–34.2	32.2	1.6
Measurements as a percentage of head length
Head depth	11.2	93.4–117.2	103.6	6.6	82.6–100.9	94.0	4.2
Head width	86.9	68.1–86.9	73.4	5.6	67.0–83.6	74.5	4.7
Snout length	15.9	9.5–18.8	15.2	4.9	9.3–17.0	14.2	2.0
Lower jaw length	31.0	20.0–31.0	24.2	3.2	15.4–25.9	20.4	2.2
Interorbital width	54.5	37.4–54.5	46.2	4.7	38.1–48.3	46.6	3.1
Eye diameter	27.6	27.2–31.6	29.4	1.7	26.6–31.4	29.3	1.4

We recorded juvenile specimens in June (2019), October (2018) and November (2017), and mature specimens in March (2018). Based on the historical climatic data for this region ( Fig. 14 ), we suspect that August and September appear to be the most likely months for drying and that the period from January to July appears to be most likely for pond filling. However, we recorded juvenile individuals (probably ~1 month after hatching) in June, October and November in different years; hence, given the climatic variability and the high general temperatures of this area, we estimate that these environments are very variable in time and that there are likely to be multiple hatching episodes and filling–drying cycles during the year in seasonal ponds from this region. In June 2019 , live juvenile individuals were netted from the type locality, measured and weighed ( Table 3 ). According to our observations, all collected specimens presented similar sizes, hence it is likely that all individuals corresponded to a single cohort from a unique hatching event or very close in time. Conservation status Following IUCN criteria ( IUCN 2012 ), the new species is considered as Vulnerable, meeting criteria D.2.: ‘Population with a very restricted area of occupancy (typically less than 20 km 2 ) or number of locations (typically five or fewer) such that it is prone to the effects of human activities or stochastic events within a very short time period in an uncertain future, and is thus capable of becoming Critically Endangered or even Extinct in a very short time period’. This species inhabits a single pond in an area that is prone to the effects of fires, climate change and exotic species such as Oreochromis niloticus (Linnaeus, 1758) that we have collected in the area, we consider it as a Vulnerable species despite being in a protected area, because these threats could damage its population severely. Behaviour The reproductive behaviour of this species in aquarium conditions appears to be like that described for other Austrolebias species ( Belote 2004 , García et al. 2008 ), as follows: (1) males present a lateral display in front of the females with deployment of unpaired fins and opening of the opercula; (2) males perform a lateral shaking of the body, pointing towards the substrate; (3) after the female pairs with the male, they burrow into the substrate; (4) the fish remain in the substrate for ~50 s, where the eggs are laid and fertilization occurs; and (5) afterwards, the fish emerge from the substrate (see Supporting Information, Appendix S10). Egg development The incubation period of the eggs at room temperature before hatching is ~2–3 months, when most eggs were observed ready to hatch. Character states proposed by Thompson et al. (2017) are coded for this species herein ( Table 4 ; Fig. 15 ). The egg size was ~ 1.3 mm . Etymology The name guarani is erected in honor of the Guaraní indigenous people, who live in several settlements very close to the type locality, as a vindication of their culture and history.