Habitat and Conservation Assessment of Annual Killifishes of the Genus Xenurolebias (Rivulidae: Cynolebiinae) from Coastal Floodplains, Including the First Record South of the Rio Doce, Southeastern Brazil^†

Response to Reviewer 1

Our responses are left in blue font.

1. Summary

Thanks so much for taking the time to review our manuscript. Please find the detailed responses below and the corresponding revisions/corrections in track changes addressed in the re-submitted files.

General comments: Molecular information is out of the scope of the studies in our laboratory, and will be left for future research.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: First, in lines 316-319 ‘Such coexistence is uncommon....’ These are not results, but discussion!

“Such coexistence is uncommon among rivulids, whose life cycle usually involves synchronization between hatching and periods of temporary flooding, with no overlap of generations” now on line 375- 377 in Round 1 version resubmitted.

We called this section Results&Discussion as suggested.

Comments 2: In lines 359-361 ‘Degradation can lead to the local extinction of the species, which highlights’

“Degradation can lead to the local extinction of the species, which highlights the need for urgent actions, such as the creation of conservation units […] now on line 320- 322 in Round 1 version resubmitted.

We called this section Results&Discussion as suggested.

Comments 3: In lines 382-384 ‘These evidences reinforce the urgent need to create protected areas’.

“These evidences reinforces the urgent need to create protected areas around the local wetlands habitats” now on line 443- 444 in Round 1 version resubmitted.

We called this section Results&Discussion as suggested.

Comments 4: the brief discussion, as currently written, should be rewritten

Results&Discussion are written together with additional information. General questions addressed to Conclusions.

Comments 5: emphasizing the urgent need to reassess the conservation status of these species and by offering concrete suggestions for conservation actions. The text clearly highlights that the current classifications are outdated and undervalued. Given this, why didn’t the authors propose updated conservation statuses for these species based on the collected data, following IUCN methodology? I don’t understand why they calculated the EOO but did not apply the classification criteria of the IUCN to suggest revised threat categories.

Conclusions adds suggestions on conservation status of two species- The DD (Data Deficient) Xenurolebias cricarensis and the new species Xenurolebias tupinikin, as follows:

Lines 553- 554- It is suggested that these fishes can be considered as Critically Endangered species in the next assessment of endangered fauna.

New figures and maps added reinforcing the AOO and EOO. New images for species Xenurolebias pataxo added, after a supplementary field trip.

Comments 6: This area is full of small independent streams’; what do the authors mean by independent? the same in line 190 ‘first independent

Explained in Lines 44- 48- As independent streams we mean small river basins flowing directly to the Ocean with no connection to a large river basin. It is a quite common condition in the landscape at Coastal Tablelands, due to almost plain relief. In mountainous river basins along Atlantic Forest a common condition is a river capture towards neighbor river basins.

Comments 7: Lines 81-83: I think that more details should be given in the study area about the hydrological dynamics of these ecosystems, namely about the relationship between lotic and lentic systems.

Explained in Lines 102- 111- The coastal tablelands between northern Espírito Santo and southern Bahia, within the larger Atlantic Forest biome, are characterized by their relatively flat surfaces, deeply incised valleys, and unconsolidated sedimentary deposits [35]. These tablelands, formed by the Barreiras Formation, are a significant source of sediment for the coastal zone and are used for agriculture, particularly coffee crops and fruits. The hydrological dynamics of these lowlands are markedly complex [36]. The lotic systems are peculiar with large river systems and also small rivers, flowing in slow current. In lowlands there are plenty lentic environments as lake systems, ponds, puddles and swamps. These areas experience variations in water levels due to precipitation and topography, leading to diverse aquatic habitats.

Comments 8: Line 101-104: Which field protocol?

We used a protocol in the field. All measurements are Explained in Line 533- Table 2.  Abiotic data for Xenurolebias species. All data were achieved during fieldwork.

Comments 9: Line 195: ‘on two tributaries’? But they weren't found in lentic zones?; rio Riacho or Riacho River?

Our mistake. Text rewritten at Line 249- the aquatic environments have predominantly sandy substrate […]

All names uniformized as Rio Riacho.

Comments 10: Lines 195-204: The English in these paragraphs needs to be improved – rewrite these sentences.

Text rewritten at Lines 247-250-Flowing along lowlands, the Rio Riacho crosses savannah formations as Restinga and Muçununga. Its aquatic environments have predominantly sandy substrate and banks covered by riparian vegetation in different states of conservation.

Comments 11: Line 230: approximately 4 km in straight line’; we can't see this in the figure - where is the Atlantic coast?

Lines 284- Information added to Figure 6 legend- Localities corresponds to those marked in map at Fig. 4.

Comments 12: Line 233: This figure should be redrawn because the colours of the river basins and the sampled sites are very confusing - as it is, it's difficult to see

Line 307- A new figure less colored added.

Comments 13: Line 265-266: but it seems that some populations persist all year round and are not associated with this dynamic... develop these aspects in the introduction.

Line 39-41- Locally, a habitat variation occurs and along coastal tablelands lowlands some pools are deeper and maintain surface water for longer periods, providing refuge for Xenurolebias populations.

Comments 14: Line 373 ‘single location’? but aren't there two locations on the map?

Line 451-453- In the Red List criteria, “location” refers to a threat-based area, and is different from the notion of locality. A location corresponds to an area where a single development project can rapidly eliminate or severely reduce the population [37].

Comments 15: Line 378 ‘thousand square metres

Line 460- padronized as m² or km²

Thanks for the constructive comments.

Response to Reviewer 2

1. Summary

Thanks so much for taking the time to review our manuscript. Please find the detailed responses below and the corresponding revisions/corrections in track changes addressed in the re-submitted files.

2. Point-by-point response to Comments and Suggestions for Authors

Comments 1: - In the abstract of the article, line 29, to replace “to highlight the occupied habitat” with “to characterize the occupied habitat”… -

Response 1: Done. Line 29- The present contribution aims to characterize the occupied habitat by each species within the temporary environments

Comments 2: In the Introduction I propose the wording “From the Rivulidae fish family, the Cynolebiinae subfamily includes annual species...”     

Response 2: Done. Line 35- From the Rivulidae fish family, the Cynolebiinae subfamily includes annual species, or cloud fish,

Comments 3: Document the current taxonomic status of the species. Are they good species, also confirmed using molecular methods?

Response 3: A molecular recognition of Xenurolebias species was already provided by Costa, Amorim (2014). Molecular information is out of the scope of the studies in our laboratory, and will be left for future research. Otherwise, the new species Xenurolebias tupinikin is recognized on the basis of morphological features, and is distinctive from its congeners as stated in its Diagnosis.

Comments 4: Study area should be structured around the following issues: Geology, Pedology, Hydrology, Climate, Vegetation, Habitats, Fauna

Response 4:

Geology- Lines 81-87- The coastal tablelands between northern Espírito Santo and southern Bahia, within the larger Atlantic Forest biome, are characterized by their relatively flat to wavy terrains, deeply incised valleys, and unconsolidated sedimentary deposits [35]. These tablelands, formed by the Barreiras Formation, are a significant source of sediment for the coastal zone and are used for agriculture, particularly coffee crops and fruits. The phytophysiog-nomy of the region is composed of ombrophylous dense forest, a formation of the Atlantic Forest.

 Pedology- Lines 87-88- The soils, are formed by sediments of the Barreiras Formation and are typically well-drained. Latosoils, sandy soils covered by dense forest and pioneer vegetation [9-10]. 

Hydrology- Lines 114-119- The hydrological dynamics of these lowlands are markedly complex [36]. The lotic systems are peculiar with large river systems and also small rivers, flowing in slow current. In lowlands there are plenty lentic environments as lake systems, ponds, puddles and swamps. These areas experience variations in water levels due to precipitation and topography, leading to diverse aquatic habitats. 

Climate- Lines 89-92- The climate in the region is Am (tropical monsoon), according to the climatic classification system (Köppen – Geiger classification). The annual average temperature is 25 °C, with averages of 28 °C in summer and 21.4 °C in winter. The average annual rainfall is 1,354 mm, with rainy season between October and April.

Vegetation- Lines 93-109- Along lowlands, these tablelands include coastal ecosystem formations of vegetation such as restinga, muçununga. The Restinga corresponds to vegetal formations over sandy deposits along coastal plains.  These environments present variable vegetation, according to proximity of sea and contains herbaceous-shrubby, arbustive or arboreal formations, including species such as Clusia spp., Vismia guianensis, and representatives of the Myrtaceae family [11,31]. These coastal plains formations also include the Muçununga environments, quite similar to the Restinga, distinguished by its soil composition with thick layers of sandy sediments, spodosols (typically acidic soils). Vegetation is also variable, ranging from grassy to herbaceous-shrubby or arboreal, including species such as Byrsonima sericea, Tapirira guianensis, and Marcetia taxifolia [,32].

Habitats- Water accumulates in flooded areas that, due to the concentration of large quantities of leaves and tannin, display a tea-like or amber coloration. In these environments the freshwater drainages are marked by small streams, flowing in slow current, puddles and temporary pools. These pools are dynamic environments, with water levels and durations varying according to rainfall and additional climate factor, resulting in highly heterogeneous and fragile ephemeral wetlands [33].  

Fauna- The temporary flooded environments support unique plant and animal communities, mostly endemic, adapted to these fluctuating conditions [5, 9].

Comments 5: Explain the terms restinga, muçununga. Indicate some plant species from these plant communities

Response 5: Done. Line 84-93- The Restinga corresponds to vegetal formations over sandy deposits along coastal plains.  These environments present variable vegetation, according to proximity of sea and contains herbaceous-shrubby, arbustive or arboreal formations, including species such as Clusia spp., Vismia guianensis, and representatives of the Myrtaceae family [11,31]. These coastal plains formations also include the Muçununga environments, quite similar to the Restinga, distinguished by its soil composition with thick layers of sandy sedi-ments,  spodosoils (typically acidic soils,). In these locations, water drainage is imperfect, favoring the formation of temporary pools. Vegetation is also variable, ranging from grassy to herbaceous-shrubby or arboreal, including species such as Byrsonima sericea, Tapirira guianensis, and Marcetia taxifolia [,32].

Comments 6: In subchapter 2.2 Can you specify How many trips were organized between 2021-2025.

Response 6: Line 123-125- In search of fish populations, 22 expeditions and three field campaigns were con-ducted between December 2021 and March 2025, including visits to the type localities of the species Xenurolebias, as well as the exploration of additional environments.

Comments 7: -What criteria did you use in sampling the fish? What type of nets did you work with (give some details)

Response 7: Line 137-140- For sampling, exclusively artisanal sieves and scoops, with standardized measurements of 90 cm x 60 cm, were used as fishing equipment. This equipment proved to be more suitable for shallow environments, allowing them to reach the bottom of the pools, resulting in more effective and accurate sampling.

Comments 8:  Give details related to the water analysis

Response 8: Information organized under Table 2.

Comments 9:  -How many specimens were examined of each species?

Response 9: Line 229- Xenurolebias tupinikin- 15 specimens- as stated in Table 1.

Xenurolebias izecksohni- 57 specimens- as stated in Supplementary material.

Xenurolebias pataxo- 14 specimens- as stated in Supplementary material S1.

Xenurolebias cricarensis- 14 specimens- Line 436- Main text.

Xenurolebias myersi- 19 specimens- as stated in Supplementary material S1.

Comments 10:  -On line 124 replace the year 2035 with 2025

Response 10: Done. Line 124- three field campaigns were conducted between December 2021 and March 2025,

Comments 11:  -In table 1 explain the meaning of n

Response 11: Done. Line 230- n = number of specimens examined.

Comments 12: Insert the micrometric bar or ruler in the other photos of the fish

Response 12: Scale bar added to images.

Comments 13: -How do you assess the status of the populations of the five species?

Response 13: We inventoried all published and our samples records for each of the species. Plotted all in the maps. Lines 157-159- Based on the information achieved we calculated Extent of Occurrence (EOO) and Area of Occupancy (AOO).   

Comments 14: --How do you assess the status of the habitats? What types of impacts have you encountered? How do you assess their intensity?

Response 14: lines 562- 568- The habitats of Xenurolebias populations are in most situations severely anthropized. Among the main reasons, precursors of these impacts, are the monoculture of Eucalyptus and sugarcane plantations, drainage of lowlands environments, including the temporary puddles, deforestation and pollution. In the last ten years, impacts have been intense, especially for populations of X. pataxo, which had its habitat drastically reduced mainly due to habitat loss. Additionally, X. cricarensis and the new species X. tupinikin, have a worrisome situation, as the populations records are limited to less than three localities.

Comments 15: ---Do you consider it appropriate to monitor fish populations and habitats in order to conserve biodiversity?

Response 15: Lines 585- 589- Monitor these fish populations in their respective habitats is even more necessary now. Because this directly implies the conservation of species and their environments. In this way, it is possible to understand the dynamics of the population, its habits and behaviors. We are working on this purpose.

4. Response to Comments on the Quality of English Language

Point 1:

Careful proofreading, and italics characters added to all species names. Minor grammar errors corrected. Thanks for reminding.