Abstract

Notholebias minimus is an endangered annual killifish endemic to the coastal plains of the State of Rio de Janeiro, Brazil. This study aimed to present new occurrences in the Atlantic Forest biome, provide unprecedented population features (body and egg size, fecundity, sexual ratio, and length-weight relationship – LWR), and compare changes in land use and coverage between 1985 and 2021 in biotopes located inside and outside protected areas. Three new occurrence localities were found in shallow temporary wetlands with acidic pH (6.4 ± 0.2) and low concentrations of dissolved oxygen (2.0 ± 0.9 mg/L). Males and females total length ranged from 11.1 to 31 mm and 11 to 26 mm, respectively. Batch fecundity ranged from 18 to 40 oocytes (24.8 ± 8.8), corresponding to oocytes with sizes between 800–1,006 µm (905 ± 56). Males were significantly larger than females (W = 2193.5, p = 0.0067), but both sexes occurred in similar proportions (p = 0.472). LWR showed positive allometry (b = 3.18). Biotopes located within protected areas exhibited higher conservation. Our discoveries expand the knowledge about habitat and population features of N. minimus and reinforce the importance of establishing protected areas for the conservation of annual fish biotopes.

Keywords:
Annual fish; Atlantic Forest biome; Conservation units; Killifish; Threatened fauna

Resumo

Notholebias minimus é um peixe anual ameaçado de extinção, endêmico das planícies costeiras do Estado do Rio de Janeiro, Brasil. Neste estudo, objetivamos apresentar novas ocorrências no bioma Mata Atlântica, fornecer características populacionais inéditas (tamanho do corpo e dos ovos, fecundidade, proporção sexual e relação peso-comprimento), e comparar mudanças no uso e cobertura do solo entre 1985 e 2021 em biótopos localizados dentro e fora de unidades de conservação. Registramos três novos locais em áreas úmidas temporárias rasas com pH ácido (6,4 ± 0,2) e baixas concentrações de oxigênio dissolvido (2,0 ± 0,9 mg/L). O comprimento total de machos e fêmeas variou de 11,1 a 31 mm e de 11 a 26 mm, respectivamente. A fecundidade do lote variou entre 18–40 oócitos (24,8 ± 8,8), correspondendo a diâmetros entre 800–1.006 µm (905 ± 56). Os machos foram significativamente maiores que as fêmeas (W = 2193,5; p = 0,0067), mas ocorreram em proporções similares (p = 0,472). A relação peso-comprimento detectou alometria positiva (b = 3,18). Biótopos localizados dentro de áreas protegidas exibiram maior preservação ambiental. Nossas descobertas ampliam o conhecimento sobre as características do habitat e da população de N. minimus e reforçam a importância do estabelecimento de áreas protegidas para a conservação dos biótopos dos peixes anuais.

Palavras-chave:
Bioma Mata Atlântica; Fauna ameaçada; Peixes anuais; Peixes das nuvens; Unidades de conservação

INTRODUCTION

Rivulidae (Cyprinodontiformes) is the ninth most specious fish family in the world with about 473 valid species (Fricke et al., 2023Fricke R, Eschmeyer WN, Van der Laan R. Eschmeyer’s catalog of fishes: genera, species, references [Internet]. San Francisco: California Academy of Science; 2023. Available from: http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp
http://researcharchive.calacademy.org/r... ), occurring between southern Florida and southeast of the province of Buenos Aires (Costa, 2011Costa WJEM. Phylogenetic position and taxonomic status of Anablepsoides, Atlantirivulus, Cynodonichthys, Laimosemion and Melanorivulus (Cyprinodontiformes: Rivulidae). Ichthyol Explor Freshw. 2011; 22(3):233–49. ; Calviño et al., 2016Calviño PA, Nadalin DO, Serio MJ, López HL. Colección ictiológica del Museo de La Plata: La familia Rivulidae. ProBiota, FCNyM, UNLP Serie Técnica y Didáctica. 2016; 36:1–21.; Loureiro et al., 2018Loureiro M, Sá RO, Serra SW, Alonso F, Lanés LEK, Volcan MV et al. Review of the family Rivulidae (Cyprinodontiformes, Aplocheiloidei) and a molecular and morphological phylogeny of the annual fish genus Austrolebias Costa, 1998. Neotrop Ichthyol. 2018; 16(3):1–20. https://doi.org/10.1590/1982-0224-20180007
https://doi.org/10.1590/1982-0224-20180... ). Brazil is home to the largest component of this rich fish family, with at least 314 species distributed across all national biomes. This high richness is proportional to the anthropic threats. Rivulidae is the family with the highest number of endangered species among all vertebrates that occur in Brazil (ICMBio, 2022Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Lista oficial de espécies da fauna e flora brasileira ameaçadas de extinção. Portaria MMA Nº 148, June 7, 2022. Brasília; 2022. Available from: https://www.icmbio.gov.br/cepsul/images/stories/legislacao/Portaria/2020/P_mma_148_2022_altera_anexos_P_mma_443_444_445_2014_atualiza_especies_ameacadas_extincao.pdf
https://www.icmbio.gov.br/cepsul/images... ). Habitat loss and fragmentation are the main threats to rivulids (Costa, 2009Costa WJEM, Leal F. Egg surface morphology in the Neotropical seasonal killifish genus Leptolebias (Teleostei: Aplocheiloidei: Rivulidae). Vertebr Zool. 2009; 59(1):25–29. https://doi.org/10.3897/vz.59.e30942
https://doi.org/10.3897/vz.59.e30942 ... ). Wetlands have been drastically destroyed, both in agricultural areas and in areas undergoing urbanization, through deforestation, drainage, and landfills (Abrantes et al., 2020Abrantes YG, Medeiros LS, Bennemann ABA, Bento DM, Teixeira FK, Rezende CF et al. Geographic distribution and conservation of seasonal killifishes (Cyprinodontiformes, Rivulidae) from the Mid-Northeastern Caatinga ecoregion, northeastern Brazil. Neotrop Biol Conserv. 2020; 15(3):301–15. https://doi.org/10.3897/neotropical.15.e51738
https://doi.org/10.3897/neotropical.15.... ; Castro, Polaz, 2020Castro RMC, Polaz CNM. Small-sized fish: the largest and most threatened portion of the megadiverse Neotropical freshwater fish fauna. Biota Neotrop. 2020; 20(1):e20180683. https://doi.org/10.1590/1676-0611-bn-2018-0683
https://doi.org/10.1590/1676-0611-bn-20... ; Guedes t al., 2020Guedes GHS, Salgado FLK, Uehara W, Ferreira DLP, Araújo FG. The recapture of Leptopanchax opalescens (Aplocheiloidei: Rivulidae), a critically endangered seasonal killifish: habitat and aspects of population structure. Zoologia. 2020; 37:1–08. https://doi.org/10.3897/zoologia.37.e54982
https://doi.org/10.3897/zoologia.37.e54... ; Drawert, 2022Drawert HA. A new species of the seasonal killifish genus Moema (Cyprinodontiformes: Rivulidae) from the Piraí watershed in the Southwest Amazon basin. Neotrop Ichthyol. 2022; 20(4):e220067. https://doi.org/10.1590/1982-0224-2022-0067
https://doi.org/10.1590/1982-0224-2022-... ). Despite this, research, funding agencies, policy, and freshwater conservation have historically neglected wetlands and focused on larger water bodies and flagship species (Guedes et al., 2023Guedes GHS, Gomes ID, Nascimento AA, Azevedo MCC, Souto-Santos ICA, Buckup PA et al. Reproductive strategy of the annual fish Leptopanchax opalescens (Rivulidae) and trade-off between egg size and maximum body length in temporary wetlands. Wetlands. 2023; 43:29. https://doi.org/10.1007/s13157-023-01680-9
https://doi.org/10.1007/s13157-023-0168... ).

Rivulidae is commonly subdivided into two major groups: annual/seasonal vs. non-annual/perennial, according to the presence or absence of resistant eggs capable of carrying out a complex process of embryonic diapause during the life cycle (Loureiro et al., 2018Loureiro M, Sá RO, Serra SW, Alonso F, Lanés LEK, Volcan MV et al. Review of the family Rivulidae (Cyprinodontiformes, Aplocheiloidei) and a molecular and morphological phylogeny of the annual fish genus Austrolebias Costa, 1998. Neotrop Ichthyol. 2018; 16(3):1–20. https://doi.org/10.1590/1982-0224-20180007
https://doi.org/10.1590/1982-0224-20180... ). Embryonic diapause allows species to live in hydrologically ephemeral habitats, such as temporary wetlands, where eggs are able to remain buried in dry substrate for months waiting for environmental triggers for hatching (Furness, 2016Furness AI. The evolution of an annual life cycle in killifish: adaptation to ephemeral aquatic environments through embryonic diapause. Biol Rev. 2016; 91(3):796–812. https://doi.org/10.1111/brv.12194
https://doi.org/10.1111/brv.12194 ... ; Ishimatsu et al., 2018Ishimatsu A, Van Mai H, Martin KLM. Patterns of fish reproduction at the interface between air and water. Integr Comp Biol. 2018; 58(6):1064–85. https://doi.org/10.1093/icb/icy108
https://doi.org/10.1093/icb/icy108 ... ). This uniqueness makes annual species “invisible” during a considerable part of their life cycle, making it difficult to map species distribution areas.

The coastal plains of the State of Rio de Janeiro, located in south-eastern Brazil, are important hotspots of annual fish diversity (Costa, 2012Costa WJEM. Delimiting priorities while biodiversity is lost: Rio’s seasonal killifishes on the edge of survival. Biodivers Conserv. 2012; 21:2443–52. https://doi.org/10.1007/s10531-012-0301-7
https://doi.org/10.1007/s10531-012-0301... ). Among these endemic species, the genus NotholebiasCosta, 2008Costa WJEM. Monophyly and taxonomy of the Neotropical seasonal killifish genus Leptolebias (Teleostei: Aplocheiloidei: Rivulidae), with the description of a new genus. Zool J Linn Soc. 2008; 153(1):147–60. https://doi.org/10.1111/j.1096-3642.2008.00380.x
https://doi.org/10.1111/j.1096-3642.200... stands out including four valid species: Notholebias minimus (Myers, 1942), N. cruzi (Costa, 1988Costa WJEM. Sistemática e distribuição do complexo de espécies Cynolebias minimus (Cyprinodontiformes, Rivulidae), com a descrição de duas espécies novas. Rev Bras Zool. 1988; 5(4):557–70. https://doi.org/10.1590/S0101-81751988000400004
https://doi.org/10.1590/S0101-817519880... ), N. fractifasciatus (Costa, 1988Costa WJEM. Sistemática e distribuição do complexo de espécies Cynolebias minimus (Cyprinodontiformes, Rivulidae), com a descrição de duas espécies novas. Rev Bras Zool. 1988; 5(4):557–70. https://doi.org/10.1590/S0101-81751988000400004
https://doi.org/10.1590/S0101-817519880... ), and N. vermiculatusCosta Amorim, 2013Costa WJEM, Amorim PF. Delimitation of cryptic species of Notholebias, a genus of seasonal miniature killifishes threatened with extinction from the Atlantic Forest of south-eastern Brazil (Cyprinodontiformes: Rivulidae). Ichthyol Explor Freshw. 2013; 24(1):63–72. . All of these species are endemic to the Brazilian Atlantic Forest biome and are threatened with extinction (ICMBio, 2018, 2022Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Livro Vermelho da Fauna Brasileira Ameaçada de Extinção: Volume VI - Peixes. Brasília: ICMBio/MMA; 2018. ). There are significant gaps in knowledge regarding the distribution, habitats, life history, and ecology of Notholebias species, as well as for most annual fish. These gaps are aggravated when considering the high number of endangered species, which should reflect a greater effort in and ex situ studies to support conservation strategies. To reduce these knowledge bottlenecks, this study has as main aims (i) to present new occurrence sites of N. minimus in the Brazilian Atlantic Forest biome, (ii) to provide unprecedented population features (individual size, fecundity and egg size, sex ratio, and length-weight ratio), and (iii) to compare anthropic impacts on land use and cover between 1985 and 2021 in temporary wetlands located inside and outside protected areas, which pose a threat to the conservation of this species.

MATERIAL AND METHODS

Sampling. Fish samplings were conducted between February and December 2022 at 23 sites distributed in five localities in the coastal drainages of Sepetiba Bay and Lagoon System of Jacarepaguá (municipalities of Seropédica and Rio de Janeiro, State of Rio de Janeiro; Tab. 1). Three localities were visited for the first time during this study: Brisas APA (Área Proteção Ambiental das Brisas), UFRRJ (Universidade Federal Rural do Rio de Janeiro), and Chaperó (Chaperó solar power plant). Two other localities with previously known distribution of N. minimus were revisited: PMN Bosque da Barra (Parque Natural Municipal Bosque da Barra) and REBIO Guaratiba (Reserva Biológica Estadual de Guaratiba). The climate is seasonal tropical, with rainy summers and dry winters (Aw climate, according to the Köppen – Geiger classification). Fish were collected with immersion nets (hand nets with an oval shape, 50 x 40 cm, 1 mm of panel mesh size). After capture, they were anesthetized with hydrochloride benzocaine (50 mg/l), euthanized and fixed in 10% formalin in situ. In the laboratory, the fish were measured (precision 0.01 cm), weighed (precision 0.001 g), and after 48 h, preserved in 70% ethanol. Biometric analyses were conducted on the same day as the capture to avoid biases associated with specimen fixation/preservation. In order to reduce the impacts of sampling on fish populations, approximately 75% of specimens were returned alive to the pools after being counted (abundance). Fish were identified and sexed according to Costa ( 1988Costa WJEM. Sistemática e distribuição do complexo de espécies Cynolebias minimus (Cyprinodontiformes, Rivulidae), com a descrição de duas espécies novas. Rev Bras Zool. 1988; 5(4):557–70. https://doi.org/10.1590/S0101-81751988000400004
https://doi.org/10.1590/S0101-817519880... , 2008Costa WJEM. Monophyly and taxonomy of the Neotropical seasonal killifish genus Leptolebias (Teleostei: Aplocheiloidei: Rivulidae), with the description of a new genus. Zool J Linn Soc. 2008; 153(1):147–60. https://doi.org/10.1111/j.1096-3642.2008.00380.x
https://doi.org/10.1111/j.1096-3642.200... , 2009aquCosta WJEM. Aplocheiloid fishes of the Brazilian Atlantic Forest: history, diversity and conservation. Rio de Janeiro: Museu Nacional; 2009.i ). Vouchers were deposited in the Ichthyological Collection of the Laboratório de Ecologia de Peixes of the Universidade Federal Rural do Rio de Janeiro (LEP–UFRRJ 2588–2593) and are available for online consultation via Global Biodiversity Information Facility – GBIF ( Araújo et al., 2023Araújo FG, Guedes G, Soares Guedes GH. Base de dados da Coleção Ictiológica do LEP-UFRRJ. Version 1.8. Sistema de Informação sobre a Biodiversidade Brasileira – SiBBr; 2023. Occurrence dataset https://doi.org/10.15468/srsucy (accessed via GBIF.org on 2023-05-19).
https://doi.org/10.15468/srsucy... ). Additional records were obtained from the bibliography (Costa, Amorim, 2013Costa WJEM, Amorim PF. Delimitation of cryptic species of Notholebias, a genus of seasonal miniature killifishes threatened with extinction from the Atlantic Forest of south-eastern Brazil (Cyprinodontiformes: Rivulidae). Ichthyol Explor Freshw. 2013; 24(1):63–72. ; Costa, 2016Costa WJEM. Inferring evolution of habitat usage and body size in endangered, seasonal Cynopoeciline killifishes from the South American Atlantic Forest through an integrative approach (Cyprinodontiformes: Rivulidae). PLoS ONE. 2016; 11(7):e0159315. https://doi.org/10.1371/journal.pone.0159315
https://doi.org/10.1371/journal.pone.01... ) and online fish collections database search at Sistema de Informação sobre a Biodiversidade Brasileira – SiBBr (www.sibbr.gov.br), SpeciesLink (www.splink.org.br), and GBIF (www.gbif.org).

To assess fecundity, ovaries from spawning females (N = 5) were removed from the visceral cavity, weighted, and kept in Gilson’s solution until a complete detachment of oocytes from epithelial and ovarian walls. Eggs were counted and measured (diameter, in μm) in a microscope LEICA TL5000 Ergo. Microanatomy of the zona pellucida was examined under scanning electron microscopy Hitachi TM1000. The bath fecundity (BF), i.e., the number of eggs produced in a single spawning batch, was established from the counting of vitellogenic oocytes (Rizzo, Bazzoli, 2020)Rizzo E, Bazzoli N. Reproduction and embryogenesis. In: Baldisserotto B, Urbinati EC, Cyrino JEP, editors. Biology and physiology of freshwater neotropical fish. UK: Academic Press; 2020. p.287–313. https://doi.org/10.1016/B978-0-12-815872-2.00013-0
https://doi.org/10.1016/B978-0-12-81587... . The relative fecundity (RF) was determined by the number of vitellogenic oocytes per body size unit (1 cm).

Physical and chemical water characteristics such as temperature (°C), dissolved oxygen (mg/L), redox potential (mV), pH, electrical conductivity (μS/cm), and turbidity (FTU) were measured using a multiprobe model Hanna HI9829. Depth (cm) was measured using centimeter rulers and a digital probe (SpeedTech SM-5) at the center of the temporary wetland (equidistant from opposite shores). Each environmental variable (physical, chemical, and depth) had the average value calculated from three replicates. The measurements were taken at two sites belonging to the same sampling locality (Chaperó, codes 11–12; Tab. 2) during the dry (June) and rainy season (December) of 2022. Therefore, the environmental data presented here may not fully express the range of variability among different occurrence habitats of the species; however, they certainly provide useful evidence of the environmental characteristics to which annual fish are exposed.

Land use and cover. To assess changes in the landscape in the fish occurrence areas, buffers were established with a radius of 250 m from the centroids of the water body where fish were caught, totaling an analyzed area of ~ 0.1963 km2. In these areas, land use and cover matrices for the years 1985 and 2021 were acquired through the Mapbiomas project (v. 7.0, https://mapbiomas.org). The classification was based on annual mosaics of Landsat satellite images, and the image classification process was carried out automatically through the use of decision tree algorithms of the Random Forest type (Souza et al., 2020Souza CM Jr., Shimbo JZ, Rosa MR, Parente LL, Alencar AA, Rudorff BFT et al. Reconstructing three decades of land use and land cover changes in Brazilian biomes with landsat archive and earth engine. Remote Sens. 2020; 12(17):2735. https://doi.org/10.3390/rs12172735
https://doi.org/10.3390/rs12172735 ... ). The classification was carried out pixel by pixel, the minimum mapped unit was equivalent to 900 m2 (30 x 30 m). A customized Spatial Reference System (SRS) was used to calculate the areas based on the Albers Projection, with parameters provided by the Instituto Brasileiro de Geografia e Estatística (IBGE). The different classes of land use and cover were grouped into two categories: natural (e.g., Forest formation, Wetlands) and anthropic (e.g., Urban Infrastructure, Pasture and Agriculture), and the rate (%) of progression or regression of anthropic cover (between 1985 and 2021) was compared between areas with different territorial policies (protected vs. unprotected areas). We included in our analyses 11 out of the 13 records (6 protected/conservation units; 5 unprotected areas) presented in Tab. 2. In two instances (codes: 10 and 13; 11 and 12; Tab. 2), the distance between the sites was less than 500 m, and to avoid buffer overlap and spatial redundancy in our analyses, we considered only one location. To address potential temporal biases of protected areas created after 1985, we observed if there were conspicuous changes in land use and cover between 1985 and the year of establishment of the protected area. We noticed that the land use and land cover matrices were similar between our lower limit (1985) and the date of creation of the conservation units. Therefore, we conducted our analyses by maintaining a standardized temporal scope of comparison of 36 years (1985–2021) for all 11 locations. All geoprocessing analyses, such as creating buffers, reprojections, transforming raster’s into polygons, calculating areas of land use and cover classes, overlays, and layer sampling were performed using QGIS software v. 3.10 A Coruña (QGIS Development Team, 2022QGIS Development Team. QGIS geographic information system. Chicago, IL: Open Source Geospatial Foundation Project; 2022. Available from: http://qgis.osgeo.org
http://qgis.osgeo.org ... ).

Statistical analyses. A Mann-Whitney-Wilcoxon test was performed to compare the differences in the total body length (TL) between males and females. A possible bias in the population sex ratio was assessed by comparing the expected rate of 1:1, and tested with a chi-square test (χ2), with a 95% of the significance level. The length-weight (W = a × TLb) relationships (LWR) based on measurements of 43 individuals (males + females) was estimated by linear regression on the transformed equation: log (W) = log (a) + b log (TL) (Le Cren, 1951Le Cren ED. The length-weight relationship and seasonal cycle in gonad weight and condition in the perch (Perca fluviatilis). J Anim Ecol. 1951; 20(2):201–19. https://doi.org/10.2307/1540
https://doi.org/10.2307/1540 ... ), where W is the body weight (g), TL is the total length (cm), a is the y-intercept, and b is the slope (Froese, 2006Froese R. Cube law, condition factor, and weight-length relationships: history, meta-analysis and recommendations. J Appl Ichthyol. 2006; 22(4):241–53. https://doi.org/10.1111/j.1439-0426.2006.00805.x
https://doi.org/10.1111/j.1439-0426.200... ). All statistical analyses were conducted in an R environment (R Development Core Team, 2022R Development Core Team. R: A language and environment for statistical computing, version 4.2.2. Vienna, Austria: R Foundation for Statistical Computing; 2022. Available from: https://www.r-project.org/
https://www.r-project.org/ ... ).

RESULTS

Three new localities of occurrence of Notholebias minimus were discovered in coastal plains draining into the Sepetiba Bay, State of the Rio de Janeiro (Tab. 2; Fig. 1). Two of the new records occurred in the Seropédica Municipality: (i) inside the campus of the UFRRJ (22°46’38.4”S 43°41’03.4”W; Tab. 2, cod. 8 and 9); and (ii) on land scheduled to receive the installation of the Chaperó solar power plant (22°48’31.0”S 43°45’51.0”W; Tab. 2, codes 11–12). The third new record occurred in the Rio de Janeiro Municipality, in the Brisas APA (22°59’29.5”S 43°39’06.8”W; Tab. 2, codes 10 and 13). In these localities, a total of 156 individuals of N. minimus (70 males, 84 females, and two juveniles with undefined sex; Fig. 2) were sampled. Two localities with the previously known distribution of the species were also revisited (code 3, REBIO de Guaratiba; code 13, PNM Bosque da Barra), however, the species was not recaptured there. Among the 23 sites inspected during the study period (Tab. 1), N. minimus was recorded in only six sites (Tab. 2). Other localities shown in Tab. 2 and Fig. 1, and not mentioned here, were not inspected.

FIGURE 1 |
Map of occurrences of Notholebias minimus in coastal plains of the State of Rio de Janeiro, Brazil. Black triangles indicate the new records in this study. Black dots, records from previous studies (e.g., Costa, Amorim, 2013; Costa, 2016). Occurrence references (codes) are available in Tab. 2.

FIGURE 2 |
Males of Notholebias minimus captured in (A) Área de Proteção Ambiental das Brisas, Rio de Janeiro Municipality, and (B) in the campus of the Universidade Federal Rural do Rio de Janeiro – UFRRJ (Seropédica Municipality). Scale bar = 4 mm.

Notholebias minimus was recorded in temporary pools typical of annual killifishes, including unshaded (Fig. 3A–B) and shaded swamps in the interior/edges of small forest fragments. Floating macrophytes were present only in unshaded swamps (Fig. 3A). For the Chaperó locality, depth (cm) varied between the dry (average ± s.d., 33 ± 19 cm) and wet (85 ± 21 cm) seasons, with swamps reaching up to 105 cm in depth (Tab. 3). Physical and chemical water characteristics indicate a pH with an acidity tendency (minimum-maximum, 6.25–6.76) and low oxygen concentrations (1.1–3.8 mg/ L; Tab. 3). Other non-annual fish species occurred in sympatry with N. minimus, such as Trichopodus trichopterus (Pallas, 1770) in the Brisas APA; Phalloceros anisophallos Lucinda, 2008, Hyphessobrycon bifasciatus Ellis, 1911, and Deuterodon hastatus (Myers, 1928) in the Seropédica Municipality (Chaperó and UFRRJ localities).

FIGURE 3 |
Temporary wetlands in the Guandu River Hydrographic Region (coastal drainages of the Sepetiba Bay, State of Rio de Janeiro, Brazil) with new occurrences of Notholebias minimus. A–B. Swamps of open vegetation in Chaperó locality, C–D. Swamps in forest fragments in the campus of the Universidade Federal Rural do Rio de Janeiro – UFRRJ, and in the Área de Proteção Ambiental das Brisas, respectively.

The chi-square test did not show significant differences in the sex ratio (1.1 female: 1 male), with both sexes being captured in similar proportions (χ2 = 0.516, p = 0.472). The body size ranged from 11.1 to 31 mm (mean ± s.d., 19.1 ± 3.9 mm TL) and 11 to 26 mm (17.5 ± 3.0 mm TL), for males and females respectively. The mean body size of males was significantly larger than females (W = 2193.5, p = 0.0067). The length-weight relationship (LWR) with sexes pooled was determined by the following equation fitted to a potential curve: Wt = 0.0099 × TL 3.18 (N = 43; Fig. 4). This equation corresponds to the logarithmic form, ln W = 4.61 + 3.18 × ln L (R2= 0.92). Notholebias minimus exhibits positive allometric growth with an exponent parameter (b) equal to 3.18 (2.89–3.46; 95% confidence interval). The total number of oocytes present in the gonads (regardless of the stage of development) of females ranged from 35 to 63 (mean 50 ± 12.3 s.d). The bath fecundity (only vitellogenic oocytes) ranged from 18 to 40 (24.8 ± 8.8), corresponding to oocytes diameter ranging from 800 to 1,006 µm (905 ± 56 µm). Relative fecundity (eggs per body size unit – 1 cm) ranged from 8.1 to 16.6 (10.9 ± 3.3). Oocytes in advanced stages of development have mushroom-like projections and polygonal grooves in the zona pellucida (Fig. 5).

Seven different classes of land use and cover were mapped in adjacent areas (radius 250 m) of N. minimus occurrences (Fig. 6). The main impacts in the species occurrence areas were mosaic of land use (28.2%; areas of agricultural use where it was not possible to distinguish between pasture and agriculture), pasture (21.7%), urban area (4.8%) and other non-vegetated areas (3.2%; areas of non-permeable surfaces such as infrastructure or mining). The locations within conservation units exhibited greater relative coverage of natural matrices (total 48%; wooded sandbank vegetation 18.9%, forest formation 14.7%, and wetlands 14.2%) compared to unprotected sites (total 29.4%; wooded sandbank vegetation 0.26%, forest formation 11.7%, and wetlands 17.2%). Protected and unprotected areas also showed opposite temporal trends (1985–2021) of changes in the landscape, while unprotected areas showed an expansion of 4% of anthropic matrices, in protected areas there was a restoration of 7.3% of natural matrices (Fig. 6).

FIGURE 4 |
Length-weight relationship of Notholebias minimus (N = 43).

FIGURE 5 |
Unfertilized eggs of Notholebias minimus, evidencing mushroom-like projections and polygonal grooves in the zona pellucida. Scale bar = 100 µm.

FIGURE 6 |
Land use and cover (%) in 11 different localities (Protected/Conservation Units vs. Unprotected) and periods (1985–2021) at areas (buffer 250 m) of occurrence of Notholebias minimus.

DISCUSSION

Notholebias minimus has a remarkably wide geographic distribution compared with other species of the genus Notholebias. Records of this species include the basins of the rivers Guandu, Guarda, Portinho, and drainages of the Lagoon System of Jacarepaguá (Costa, 1988Costa WJEM. Sistemática e distribuição do complexo de espécies Cynolebias minimus (Cyprinodontiformes, Rivulidae), com a descrição de duas espécies novas. Rev Bras Zool. 1988; 5(4):557–70. https://doi.org/10.1590/S0101-81751988000400004
https://doi.org/10.1590/S0101-817519880... ; Costa, Amorim, 2013Costa WJEM, Amorim PF. Delimitation of cryptic species of Notholebias, a genus of seasonal miniature killifishes threatened with extinction from the Atlantic Forest of south-eastern Brazil (Cyprinodontiformes: Rivulidae). Ichthyol Explor Freshw. 2013; 24(1):63–72. ). This contrasts with the other species of the genus, which have lesser wide distribution and are restricted to the surroundings of the type localities (Costa, 1988Costa WJEM. Sistemática e distribuição do complexo de espécies Cynolebias minimus (Cyprinodontiformes, Rivulidae), com a descrição de duas espécies novas. Rev Bras Zool. 1988; 5(4):557–70. https://doi.org/10.1590/S0101-81751988000400004
https://doi.org/10.1590/S0101-817519880... ; Costa, Amorim, 2013Costa WJEM, Amorim PF. Delimitation of cryptic species of Notholebias, a genus of seasonal miniature killifishes threatened with extinction from the Atlantic Forest of south-eastern Brazil (Cyprinodontiformes: Rivulidae). Ichthyol Explor Freshw. 2013; 24(1):63–72. ; ICMBio, 2018Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Livro Vermelho da Fauna Brasileira Ameaçada de Extinção: Volume VI - Peixes. Brasília: ICMBio/MMA; 2018. ). There are alternative historical scenarios for the modern distribution patterns of Rivulidae (e.g., Garcia et al., 2012García G, Gutiérrez V, Vergara J, Calviño P, Duarte A, Loureiro M. Patterns of population differentiation in annual killifishes from the Paraná–Uruguay–La Plata Basin: the role of vicariance and dispersal. J Biogeogr. 2012; 39(9):1707–19. https://doi.org/10.1111/j.1365-2699.2012.02722.x
https://doi.org/10.1111/j.1365-2699.201... ; Costa et al., 2017Costa WJEM, Amorim PF, Mattos JLO. Molecular phylogeny and timing of diversification in South American Cynolebiini seasonal killifishes. Mol Phylogenet Evol. 2017; 116:61–68. https://doi.org/10.1016/j.ympev.2017.07.020
https://doi.org/10.1016/j.ympev.2017.07... ; Loureiro et al., 2018Loureiro M, Sá RO, Serra SW, Alonso F, Lanés LEK, Volcan MV et al. Review of the family Rivulidae (Cyprinodontiformes, Aplocheiloidei) and a molecular and morphological phylogeny of the annual fish genus Austrolebias Costa, 1998. Neotrop Ichthyol. 2018; 16(3):1–20. https://doi.org/10.1590/1982-0224-20180007
https://doi.org/10.1590/1982-0224-20180... ), and at smaller spatial scales, there is evidence that some species could be dispersed by rearrangements of river drainages, large floods or even endozoochory (Costa, 2013Costa WJEM. Historical biogeography of aplocheiloid killifishes (Teleostei: Cyprinodontiformes). Vertebr Zool. 2013; 63(2):139–54. https://doi.org/10.3897/vz.63.e31419
https://doi.org/10.3897/vz.63.e31419 ... ; Silva et al., 2019Silva GG, Weber V, Green AJ, Hoffmann P, Silva VS, Volcan MV et al. Killifish eggs can disperse via gut passage through waterfowl. Ecology. 2019; 100(11):e02774. https://doi.org/10.1002/ecy.2774
https://doi.org/10.1002/ecy.2774 ... ). Therefore, the explanation for the current distribution of Notholebias species is not trivial and deserves further specific studies, as they may encompass unique phylogeographic patterns.

The new biotopes were located inside shaded forest fragments and in swamps of open vegetation exposed to the sun, typical of Notholebias spp., which may still include sandy coastal areas covered by bush, grass and open woodland vegetation located up to 100 m from the sea (Costa, 1988Costa WJEM. Sistemática e distribuição do complexo de espécies Cynolebias minimus (Cyprinodontiformes, Rivulidae), com a descrição de duas espécies novas. Rev Bras Zool. 1988; 5(4):557–70. https://doi.org/10.1590/S0101-81751988000400004
https://doi.org/10.1590/S0101-817519880... ). The water in temporary pools at Chaperó locality showed an acidity tendency and low oxygen concentrations, typical environmental conditions of temporary wetlands (Bidwell, 2013,Bidwell JR. Physical and chemical monitoring of wetland water. In: Anderson J, Davis C, editors. Wetland Techniques. Dordrecht: Springer; 2013. p.325–53. https://doi.org/10.1007/978-94-007-6860-4_6
https://doi.org/10.1007/978-94-007-6860... ). Overall, annual killifish have evolved to withstand significant daily and seasonal environmental changes, including variations in temperature, oxygen concentration, salinity, pH, and water availability, that approach the limits of vertebrate survival (Podrabsky et al., 2016Podrabsky JE, Riggs CL, Wagner JT. Tolerance of environmental stress. In: Berois N, García G, Sá RO, editors. Annual fishes: life history strategy, diversity, and evolution. Boca Ratón: CRC Press; 2016. p.160–80. ; Polačik, Podbrabsky, 2016Polačik M, Podbrabsky JE. Temporary environments. In: Riesch R, Plath M, Tobler M, editors. Extremophile fishes – Ecology and evolution of teleosts in extreme environments. New York: Springer; 2016. p.217–45. https://www.springer.com/gp/book/9783319133614
https://www.springer.com/gp/book/978331... ; Ishimatsuet al., 2018Ishimatsu A, Van Mai H, Martin KLM. Patterns of fish reproduction at the interface between air and water. Integr Comp Biol. 2018; 58(6):1064–85. https://doi.org/10.1093/icb/icy108
https://doi.org/10.1093/icb/icy108 ... ). The co-occurrence between N. minimus and other non-annual species (T. trichopterus, P. anisophallos, H. bifasciatus, D. hastatus) indicates a periodic connection of the temporary wetlands with adjacent perennial water bodies. Sympatry between Notholebias and other annual and non-annual species is common (Costa, 1988Costa WJEM. Sistemática e distribuição do complexo de espécies Cynolebias minimus (Cyprinodontiformes, Rivulidae), com a descrição de duas espécies novas. Rev Bras Zool. 1988; 5(4):557–70. https://doi.org/10.1590/S0101-81751988000400004
https://doi.org/10.1590/S0101-817519880... ; ICMBio, 2018Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Livro Vermelho da Fauna Brasileira Ameaçada de Extinção: Volume VI - Peixes. Brasília: ICMBio/MMA; 2018. ) and indicates that these species are able to complete their life cycle and maintain viable populations even under periodic competition or predation.

Notholebias minimus showed a positive allometric growth (b = 3.18), with comparatively more gain in weight than in length (Froese, 2006Froese R. Cube law, condition factor, and weight-length relationships: history, meta-analysis and recommendations. J Appl Ichthyol. 2006; 22(4):241–53. https://doi.org/10.1111/j.1439-0426.2006.00805.x
https://doi.org/10.1111/j.1439-0426.200... ). However, no previous references were found for the LWR of N. minimus and other species of Notholebias, what prevents comparisons of our results with other studies. Males of N. minimus are larger than females, corroborating the pattern of sexual dimorphism commonly observed in other species of Rivulidae (e.g., Arenzon et al., 2001Arenzon A, Peret AC, Bohrer MBC. Growth of the annual fish Cynopoecilus melanotaenia (Regan, 1912) based in a temporary water body population in Rio Grande do Sul State, Brazil (Cyprinodontiformes, Rivulidae). Braz J Biol. 2001; 61(1):117–23. https://doi.org/10.1590/s0034-71082001000100015
https://doi.org/10.1590/s0034-710820010... ; Lanés et al., 2012Lanés LEK, Keppeler FW, Maltchik L. Abundance, sex-ratio, length-weight relation, and condition factor of non-annual killifish Atlantirivulus riograndensis (Actinopterygii: Cyprinodontiformes: Rivulidae) in Lagoa do Peixe National Park, a Ramsar Site of Southern Brazil. Acta Ichthyol Piscat. 2012; 42(3):277–52. https://doi.org/10.3750/AIP2011.42.3.09
https://doi.org/10.3750/AIP2011.42.3.09... ; Guedes et al., 2020Guedes GHS, Salgado FLK, Uehara W, Ferreira DLP, Araújo FG. The recapture of Leptopanchax opalescens (Aplocheiloidei: Rivulidae), a critically endangered seasonal killifish: habitat and aspects of population structure. Zoologia. 2020; 37:1–08. https://doi.org/10.3897/zoologia.37.e54982
https://doi.org/10.3897/zoologia.37.e54... ). Preparation for reproduction can cause oxidative stress and affect maternal self-maintenance (Godoy et al., 2020Godoy RS, Lanés LEK, Castro BD, Weber V, Wingen N, Pires MM et al. Oxidative stress resistance in a short-lived Neotropical annual killifish. Biogerontology. 2020; 21:217–29. https://doi.org/10.1007/s10522-019-09855-w
https://doi.org/10.1007/s10522-019-0985... ) and consequently the somatic growth of females. Differences in body size mediate the coexistence of annual fish in temporary pools by mitigating intra and interspecific competition (Arenzon et al., 2001Arenzon A, Peret AC, Bohrer MBC. Growth of the annual fish Cynopoecilus melanotaenia (Regan, 1912) based in a temporary water body population in Rio Grande do Sul State, Brazil (Cyprinodontiformes, Rivulidae). Braz J Biol. 2001; 61(1):117–23. https://doi.org/10.1590/s0034-71082001000100015
https://doi.org/10.1590/s0034-710820010... ; Volcan et al., 2019aquiVolcan MV, Gonçalves AC, Guadagnin DL. Body size and population dynamics of annual fishes from temporary wetlands in Southern Brazil. Hydrobiologia. 2019; 827:367–78. https://doi.org/10.1007/s10750-018-3789-3
https://doi.org/10.1007/s10750-018-3789... ). Therefore, intraspecific differences observed in body size between males and females may be associated with different reproductive energy costs, in addition to playing an important role in population coexistence.

A reduced batch fecundity (24.8 ± 8.8 eggs) was found for N. minimus, as well as for other annual species such as Cynopoecilus melanotaenia (Regan, 1912) (19 ± 26 eggs; Gonçalves et al., 2011Gonçalves CS, Souza UP, Volcan MV. The opportunistic feeding and reproduction strategies of the annual fish Cynopoecilus melanotaenia (Cyprinodontiformes: Rivulidae) inhabiting ephemeral habitats on southern Brazil. Neotrop Ichthyol. 2011; 9(1):191–200. https://doi.org/10.1590/S1679-62252011000100019
https://doi.org/10.1590/S1679-622520110... ), Austrolebias nigrofasciatus Costa & Cheffe, 2001 (21.5 ± 12 eggs; Volcan et al., 2011Volcan MV, Fonseca AP, Robaldo RB. Reproduction of the threatened Annual Killifish Austrolebias nigrofasciatus (Cyprinodontiformes: Rivulidae), confined in a natural environment. J Threat Taxa. 2011; 3(6):1864–67. https://doi.org/10.11609/JoTT.o2575.1864-7
https://doi.org/10.11609/JoTT.o2575.186... ), and Leptopanchax opalescens (Myers, 1942) (27 ± 7.0 eggs; Guedes et al., 2023Guedes GHS, Gomes ID, Nascimento AA, Azevedo MCC, Souto-Santos ICA, Buckup PA et al. Reproductive strategy of the annual fish Leptopanchax opalescens (Rivulidae) and trade-off between egg size and maximum body length in temporary wetlands. Wetlands. 2023; 43:29. https://doi.org/10.1007/s13157-023-01680-9
https://doi.org/10.1007/s13157-023-0168... ). However, the eggs are relatively large (maximum 1.006 μm) when weighted by the spatial limitations imposed by the coelomic cavity in this species of reduced body size (< 4 cm). According to the optimal egg size theory, populations evolve a particular egg size that balances the tradeoff between egg size and fecundity to maximize reproductive yield (Smith, Fretwell, 1974Smith CC, Fretwell SD. The optimal balance between size and number of offspring. Am Nat. 1974; 108(962):499–506. https://doi.org/10.1086/282929
https://doi.org/10.1086/282929 ... ). Therefore, larger eggs come at a cost of reducing the number of eggs, which is in accordance with the findings of this study. Annual species have smaller eggs when compared to non-annual species of the family Rivulidae ( Guedes et al., 2023 Guedes GHS, Gomes ID, Nascimento AA, Azevedo MCC, Souto-Santos ICA, Buckup PA et al. Reproductive strategy of the annual fish Leptopanchax opalescens (Rivulidae) and trade-off between egg size and maximum body length in temporary wetlands. Wetlands. 2023; 43:29. https://doi.org/10.1007/s13157-023-01680-9
https://doi.org/10.1007/s13157-023-0168... ). This may be associated with the extreme tolerance of embryos to hypoxia due to the process of embryonic diapause, which culminates in developmental arrest, metabolic depression, and G1 cell cycle arrest ( Podrabsky et al., 2016Podrabsky JE, Riggs CL, Wagner JT. Tolerance of environmental stress. In: Berois N, García G, Sá RO, editors. Annual fishes: life history strategy, diversity, and evolution. Boca Ratón: CRC Press; 2016. p.160–80. ). For species without embryonic diapause, the optimal investment in offspring size increases as environmental quality decreases ( Rollinson, Hutchings, 2013Rollinson N, Hutchings JA. Environmental quality predicts optimal egg size in the wild. Am Nat. 2013; 182(1):76–90. https://doi.org/10.1086/670648
https://doi.org/10.1086/670648 ... ; Riesch et al., 2014Riesch R, Plath M, Schlupp I, Tobler M, Brian LR, Gaillard JM. Colonization of toxic environments drives predictable life-history evolution in live-bearing fishes (Poeciliidae). Ecol Lett. 2014;17(1):65–71. https://doi.org/10.1111/ele.12209
https://doi.org/10.1111/ele.12209 ... ; Santi et al., 2021Santi F, Vella E, Jeffress K, Deacon A, Riesch R. Phenotypic responses to oil pollution in a poeciliid fish. Environ Pollut. 2021; 290:118023. https://doi.org/10.1016/j.envpol.2021.118023
https://doi.org/10.1016/j.envpol.2021.1... ). The zona pellucida of mature eggs of N. minimus featured mushroom-like projections similar to other species in the genera Leptopanchax and Notholebias (Costa, Leal, 2009Costa WJEM, Leal F. Egg surface morphology in the Neotropical seasonal killifish genus Leptolebias (Teleostei: Aplocheiloidei: Rivulidae). Vertebr Zool. 2009; 59(1):25–29. https://doi.org/10.3897/vz.59.e30942
https://doi.org/10.3897/vz.59.e30942 ... ; Thompson et al., 2017 Thompson AW, Furness AI, Stone C, Rade CM, Ortí G. Microanatomical diversification of the zona pellucida in aplochelioid killifishes. J Fish Biol. 2017; 91(1):126–43. https://doi.org/10.1111/jfb.13332
https://doi.org/10.1111/jfb.13332 ... ). Wourms, Sheldon (1976)Wourms JP, Sheldon H. Annual fish oogenesis: II. Formation of the secondary egg envelope. Dev Biol. 1976; 50(2):355–66. https://doi.org/10.1016/0012-1606(76)90157-3
https://doi.org/10.1016/0012-1606(76)90... hypothesized that these projections are a chorionic respiratory system since there is a network of channels leading to hollow spikes that may function as egg-like aeropiles, similar to insect eggs. This may be an adaptation for annual fishes since a thick, hard, and consequently poorly oxygen-permeable zona pellucida may be necessary to prevent desiccation ( Thompson et al., 2017Thompson AW, Furness AI, Stone C, Rade CM, Ortí G. Microanatomical diversification of the zona pellucida in aplochelioid killifishes. J Fish Biol. 2017; 91(1):126–43. https://doi.org/10.1111/jfb.13332
https://doi.org/10.1111/jfb.13332 ... ).

Notholebias minimus is currently found in five conservation units in the State of Rio de Janeiro, including the unpublished record in the Brisas APA presented here. However, other species such as Notholebias vermiculatus and N. fractifasciatus do not occur in protected areas (ICMBio, 2018Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). Livro Vermelho da Fauna Brasileira Ameaçada de Extinção: Volume VI - Peixes. Brasília: ICMBio/MMA; 2018. ). Notholebias cruzi whose type locality is outside a conservation unit, had its biotopes destroyed due to urban expansion and has not been found since 2002, and may be extinct (Costa, 2012Costa WJEM. Delimiting priorities while biodiversity is lost: Rio’s seasonal killifishes on the edge of survival. Biodivers Conserv. 2012; 21:2443–52. https://doi.org/10.1007/s10531-012-0301-7
https://doi.org/10.1007/s10531-012-0301... ; Lira, 2021Lira FO. A retórica da perda da Mata Atlântica nos lamentos dos peixes das nuvens ou do nosso dilema do Tilacino. O ECO; 2021. ). Biotopes of N. minimus located inside conservation units show great natural cover and environmental restoration trends between 1985 and 2021. On the other hand, locations without any protection show greater coverage of anthropic matrices (pasture, urban area) and a loss of temporary wetlands between 1985 and 2021. These results show the important role played by protected areas in the conservation of biotopes. However, even the protected areas showed high coverage (52%) of anthropic matrices, which may reflect the type of territorial policy, since part of these units are for sustainable use and consequently have fewer restrictions on land use (SNUC, 2000Sistema Nacional de Unidades de Conservação da Natureza (SNUC). Sistema Nacional de Unidades de Conservação da Natureza. Lei n° 9.985, July 18, 2000. Brasília; 2000. Available from: https://www.planalto.gov.br/ccivil_03/leis/l9985.htm
https://www.planalto.gov.br/ccivil_03/l... ), and/or historical deforestation prior to 1985, since the Brazilian Atlantic Forest biome is historically impacted (Joly et al., 2014Joly CA, Metzger JP, Tabarelli M. Experiences from the Brazilian Atlantic Forest: ecological findings and conservation initiatives. New Phytol. 2014; 204(3):459–73. https://doi.org/10.1111/nph.12989
https://doi.org/10.1111/nph.12989 ... ; Egler et al., 2020Egler C, Nielsen D, Lira FO, Gusmão F. A expansão urbana do Rio de Janeiro e o peixe das nuvens. Rio de Janeiro: Andrea Jakobsson Estúdio; 2020. ).

The wide geographic distribution of N. minimus, combined with records in conservation units, places this species in a more favorable conservation position when compared to other species of the genus Notholebias. Our findings reveal that biotopes located within protected areas show a trend of restoration between 1985–2021, with an advancement of natural matrices. Conversely, biotopes found in unprotected areas show an opposite trend, with an increase in anthropogenic impacts on land use and coverage. However, it is crucial to maintain continuous monitoring of the biotopes, both inside and outside protected areas, to ensure the successful preservation of these endangered fish. In conclusion, our findings expand the knowledge of the habitats and population structure of N. minimus, and reinforce the importance of establishing protected areas for the conservation and restoration of annual fish biotopes.

ACKNOWLEDGEMENTS

This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Proc. #140512/2022–5; 305712/2020–9; 306792/2021–4), Fundação Carlos Chagas Filho de Amparo à Pesquisa no Estado do Rio de Janeiro – FAPERJ (Proc. E–26/200.897/2021; E–26/202.483/2021), Fundo Brasileiro para a Biodiversidade – FUNBIO Conservando o Futuro, and Instituto HUMANIZE (Proc. # 028/2023). Special thanks to Yuri Borba for photographing the fish and habitat at Área de Proteção Ambiental das Brisas.

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