Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Environmental DNA exploration of fish diversity

in the Balantak River, Central Sulawesi, Indonesia

Magdalena Litaay1*; https://orcid.org/0000-0002-7743-0960

Asmi Citra Malina Tassakka2; https://orcid.org/0000-0001-8967-210X

Nadiarti Nurdin Kadir2,9; https://orcid.org/0000-0003-2837-1914

Widyastuti Umar3,9; https://orcid.org/0000-0003-0763-6581

Samliok Ndobe4,9; https://orcid.org/0000-0003-4221-5205

Abdul Gani5,8,9; https://orcid.org/0000-0001-9730-4251

Arfiani Rizki Paramata6,9; https://orcid.org/0000-0002-9619-2494

Nur Afifah Zhafirah1; https://orcid.org/0009-0005-9040-386X

Abigail Mary Moore7,9; https://orcid.org/0000-0002-4122-3740

1. Department of Biology, Faculty of Mathematics and Natural Sciences, Hasanuddin University, Makassar, South

Sulawesi, Indonesia; mlitaay@fmipa.unhas.ac.id (*Correspondence), fifah.zhafirah@gmail.com

2. Department of Fisheries, Faculty of Marine Science and Fisheries, Hasanuddin University, Makassar, South Sulawesi,

Indonesia; citra@unhas.ac.id, nadiarti@unhas.ac.id

3. Department of Marine Science, Faculty of Marine Science and Fisheries, Hasanuddin University, Makassar, South

Sulawesi, Indonesia; widyastuti@unhas.ac.id

4. Fisheries and Marine Department, Tadulako University, Palu, Central Sulawesi, Indonesia; samliok@untad.ac.id

5. Aquaculture Study Program, Faculty of Fisheries, Universitas Muhammadiyah Luwuk, Banggai Regency, Central

Sulawesi, Indonesia; abdulgani273085@gmail.com

6. Fishery Resources Management Department, Faculty of Marine Science and Fisheries Technology, Gorontalo State

University, Gorontalo, Indonesia; arfiani@ung.ac.id

7. Graduate School, Hasanuddin University, Makassar, South Sulawesi, Indonesia; abigail@pasca.unhas.ac.id

8. Doctoral Program in Fisheries Science, Faculty of Marine Science and Fisheries, Hasanuddin University, Makassar,

South Sulawesi, Indonesia; abdulgani273085@gmail.com

9. Aquatic Macrofauna Biodiversity and Conservation Research Group, Hasanuddin University, Makassar, South

Sulawesi, Indonesia; abdulgani273085@gmail.com, abigail@pasca.unhas.ac.id, arfiani@ung.ac.id, nadiarti@unhas.

ac.id, samliok@untad.ac.id, widyastuti@unhas.ac.id

Received 11-XI-2025. Corrected 22-III-2026. Accepted 17-V-2026.

ABSTRACT

Introduction: Freshwater ecosystems play a crucial role in supporting human life, providing many ecosystem

services. Species diversity is a valuable indicator for evaluating ecosystem health, gaining a broader perspective

on ecological richness and ecosystem functions, but data are lacking for many rivers.

Objectives: To obtain baseline data on the upstream and downstream ichthyofauna of the Balantak River,

Banggai Regency, using environmental DNA (eDNA) with a focus on diadromous species.

Methods: We collected, filtered and preserved water samples from upstream and downstream sites on the

Balantak River followed by eDNA analysis (DNA extraction, PCR with MiFish 12S rRNA primers, Illumina

sequencing, MitoFish bioinformatic analysis at 97 and 99 % identity thresholds) to produce taxonomically

assigned Actinopteri amplicon sequence variants (ASVs).

https://doi.org/10.15517/hp29s516

AQUATIC ECOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

INTRODUCTION

Freshwater ecosystems cover a small frac-

tion of the earth’s surface, with estimates vary-

ing from around 0.5 % (Ottoni et al., 2023) to

2 % (Reid et al., 2019), but they provide habitat

for around 40 % of fishes, with around 10 000-

15 000 species described to date (Reid et al.,

2019; Tedesco et al., 2017; Wang et al., 2024).

Freshwater ecosystems are among the most

valuable in terms of ecosystem services of direct

value to humans as well as intrinsic and option

values (Albert et al., 2021; Dudgeon, 2020;

Lynch et al., 2020; Reid et al., 2019). Freshwater

fisheries are considered essential to meeting

the Sustainable Development Goals (SDGs),

Results: Our data are consonant with the expected dominance of diadromous and euryhaline taxa (75 %),

high Gobiiformes biodiversity, and differences between upstream and downstream ichthyofaunal communi-

ties. Reads were assigned to Actinopteri ASVs from 12 orders, predominantly Gobiiformes, Anguilliformes,

and Centrarchiformes. The 15 assigned families included Anguillidae, Eleotridae, Gobiidae, Rhyacichthydae,

Kuhliidae, and Terapontidae. Of the assigned genera (17 at 97 % and 25 at 99 %), Anguilla had the most reads

(39.9 %), and Sicyopterus had the most ASVs (8).

Conclusions: These baseline data demonstrate the usefulness of eDNA metabarcoding for exploring ichthyofau-

nal biodiversity in small tropical watersheds while highlighting gaps in 12S rRNA sequence databases, reinforc-

ing the need for integrative taxonomic research in Sulawesi watersheds and reliable reference databases. Most

assigned taxa are vulnerable to exploitation for food and/or the ornamental fish trade. Four non-native taxa from

Cichlidae, Cyprinidae and Clariidae are considered invasive. Implications for conservation include the need to

address the unregulated and largely unreported freshwater ornamental trade and introduction of non-native

invasive species.

Key words: amphidromy; Gobiiformes; metabarcoding; riverine biodiversity; Wallacea.

RESUMEN

Exploración del ADN ambiental de la diversidad de peces

en el río Balantak, Sulawesi Central, Indonesia

Introducción: Los ecosistemas de agua dulce desempeñan un papel crucial en el mantenimiento de la vida

humana, al proveer numerosos servicios ecosistémicos. La diversidad de especies constituye un indicador valioso

para evaluar la salud de los ecosistemas, obtener una perspectiva más amplia y conservar la riqueza ecológica y

las funciones ecosistémicas; sin embargo, faltan datos para muchos ríos.

Objetivos: Obtener datos de referencia sobre la ictiofauna de los tramos alto y bajo del río Balantak, en la regencia

de Banggai, mediante ADN ambiental (eDNA) con énfasis en especies diádromas.

Métodos: Se recolectaron, filtraron y preservaron muestras de agua en sitios aguas arriba y aguas abajo del río

Balantak, seguidas de análisis de eDNA (extracción de ADN, PCR con cebadores MiFish 12S rRNA, secuenciación

Illumina, análisis bioinformático con MitoFish a umbrales de identidad de especie del 97 y 99 %) para generar

variantes de secuencia de amplicones (ASVs) de Actinopteri con asignación taxonómica.

Resultados: Nuestros datos confirman el dominio esperado de taxones diádromos y eurihalinos (75 %), una alta

biodiversidad de Gobiiformes y diferencias entre las comunidades ictiofaunales de los tramos alto y bajo del río.

Las lecturas se asignaron a ASVs de Actinopteri pertenecientes a 12 órdenes, predominantemente Gobiiformes,

Anguilliformes y Centrarchiformes. Las 15 familias asignadas incluyeron Anguillidae, Eleotridae, Gobiidae,

Rhyacichthyidae, Kuhliidae y Terapontidae. Entre los géneros identificados (17 al 97 % y 25 al 99 %), Anguilla

presentó el mayor número de lecturas (39.9 %), mientras que Sicyopterus tuvo el mayor número de ASVs (8).

Conclusiones: Se demuestra la utilidad del metabarcoding de eDNA para explorar la biodiversidad ictiofaunística

en pequeñas cuencas tropicales, y resalta vacíos en las bases de datos de secuencias 12S rRNA, reforzando la nece-

sidad de investigación taxonómica integrativa en las cuencas hidrográficas de Sulawesi y de bases de datos de refe-

rencia confiables. La mayoría de los taxones asignados son vulnerables a la explotación para consumo y/o como

peces ornamentales, y cuatro taxones exóticos pertenecientes a Cichlidae, Cyprinidae y Clariidae se consideran

invasores. Las implicaciones para la conservación incluyen la necesidad de abordar el comercio ornamental de

agua dulce no regulado y en gran medida no reportado, así como la introducción de especies exóticas invasoras.

Palabras clave: anfidromía; Gobiiformes; metabarcodificación; biodiversidad fluvial; Wallacea.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

especially SDG 1 (No Poverty), SDG 2 (Zero

Hunger), and SDG 15 (Life on Land) (Lynch

et al., 2020). While freshwater ecosystems fall

primarily under SDG 15, and the primary

scope of SDG 14 comprises oceans, seas, and

marine resources, SDG 14 and SDG 15 are

both directly relevant for many aquatic taxa,

in particular diadromous and euryhaline fishes

(Hansen et al., 2023).

Despite growing awareness of their impor-

tance, many freshwater fishes and their habitats

are imperiled, with threats growing in terms

of their variety and severity (Albert et al.,

2021). Key threats include exploitation as food

and ornamental fish, the introduction of non-

native species, direct and indirect impacts of

habitat alteration (e.g. pollution, flow regula-

tion, land-use change), local impacts of global

climate change, and often synergistic interac-

tions among multiple threats (Dudgeon, 2019;

Nurjirana et al., 2024; Oikonomou, 2021; Reid

et al., 2019; Sorensen, 2021). Extinction rates

are considerably higher for freshwater taxa

than for most other groups (Ottoni et al., 2023;

Williams-Subiza & Epele, 2021). Accurate and

comprehensive biodiversity data are vital for

understanding ecology, designing appropriate

and effective conservation policies, assessing

the impacts of threats and the effectiveness of

actions taken to address the issues identified

(Maasri et al., 2022; Oikonomou, 2021; Ottoni

et al., 2023). However, despite considerable

progress in recent decades, much remains to

be discovered in terms of species richness,

functional diversity, evolutionary patterns, and

biogeographical patterns across multiple spatial

and temporal scales (Dudgeon, 2020; Maasri et

al., 2022; Oikonomou, 2021).

Indonesia, the largest archipelagic nation,

spans three major bioregions (Sundaland, Wal-

lacea, and the Sahul Shelf) and is recognized

as a mega-biodiversity country (Lohman et al.,

2011). Sulawesi, the largest island in the Walla-

cea bioregion, is renowned for its diverse aquat-

ic biota, including high levels of endemism

(Ambo-Rappe & Moore, 2019; Lohman et al.,

2011). Miesen et al. (2016) listed 226 fishes

from 112 genera and 56 families that have been

reported from, or are likely to inhabit, Sulawesi

freshwater habitats, including primary freshwa-

ter and diadromous species. The latter include

catadromous species such as freshwater eels

(genus Anguilla) and amphidromous species,

including many Gobiiformes, which together

contribute most of the riverine ichthyofau-

nal diversity (Wantania et al., 2024). Ongoing

discoveries and new species records continue

to enhance our understanding of the Sulawesi

ichthyofauna (Gani, Nurjirana et al., 2021;

Keith, Hadiaty et al., 2014; Ndobe et al., 2022;

Nurjirana et al., 2024; Wantania et al., 2024).

However, the Sulawesi primary freshwater

and diadromous biota, including fish, are still

understudied, raising the concern that aquatic

populations, species and communities could be

depleted or lost before they are even recorded

(Ndobe et al., 2022; (Ndobe et al., 2023; Nur-

jirana et al., 2024). Furthermore, invasive alien

species have been recorded in several Sulawesi

lakes and rivers (Herder et al., 2012; Herder

et al., 2022; Paramata et al., 2025; Serdiati et

al., 2020; Yanuarita et al., 2020). These studies

highlight the need for further exploration in

Sulawesi freshwater habitats, to fill the many

gaps in our knowledge of the species present

and their distributions. Such benchmarks are

important not only to document the native spe-

cies present but also to determine which species

are truly non-native and to document their

arrival and spread.

Addressing such gaps is important from

scientific, conservation, fisheries, and environ-

mental management viewpoints. Biodiversity

is increasingly recognized as vital for resil-

ience and sustainability, now and in the face

of change (Bartholomew et al., 2024; Oliver et

al., 2015). Data on fish species, including their

distribution and status, are vital for resource

management, in conservation, fisheries, and

sustainable development, and baselines are vital

for planning and monitoring progress (Albert

et al., 2021; Bartholomew et al., 2024; Ojaveer et

al., 2018). For example, the World Bank (under

Standard ESS6) and other funding agencies

increasingly mandate biodiversity assessments

and action plans for all funded projects (Morley

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

et al., 2021; Nedopil et al., 2024). Furthermore,

aquatic biodiversity monitoring can provide

valuable indicators to assess ecosystem health

(Robinson et al., 2022).

The exploration of biodiversity in fresh-

water and other ecosystems can be conducted

using a variety of approaches, individually or

in combination. Classic taxonomic surveys are

now often combined with molecular methods

such as DNA barcoding and environmental

DNA (eDNA) metabarcoding (Chen et al.,

2024; Iqram et al., 2024; Shu et al., 2021). Each

of these approaches has strengths and limita-

tions in terms of detection and identification

(McElroy et al., 2020). Despite the advantages

of classical surveys, such as enabling the col-

lection of reference specimens, logistics, sam-

pling gear type, seasonality and fish behavior

are some of the many reasons why taxa can be

missed when collecting specimens for classical

and DNA-based identification (McElroy et al.,

2020; Wantania et al., 2024). Although eDNA

metabarcoding suffers from some of these limi-

tations, in particular with respect to seasonality,

one key advantage is that it can be considered

a non-invasive method as it does not require

direct observation or capture (Chen et al., 2024;

Hidayani et al., 2024; Wang et al., 2024). The

capacity to enable rapid identification, and even

generate occurrence records, can be particu-

larly advantageous in situations where collec-

tion could be dangerous, difficult, expensive, or

threaten sensitive ecosystems and endangered

taxa, including the detection and monitoring of

endangered species or, conversely, non-native

and potentially invasive species (Chen et al.,

2024; Shu et al., 2021). It is therefore not sur-

prising that eDNA methods are increasingly

used for aquatic biodiversity surveys and moni-

toring, including both preliminary or baseline

surveys and comprehensive biodiversity assess-

ments across diverse ecosystems and taxonomic

groups (Chen et al., 2024; McElroy et al., 2020;

Porter & Hajibabaei, 2018; Ruppert et al., 2019).

A recent review of the literature on aquatic

eDNA (Takahashi et al., 2023) found the ten

most used primer sets targeted four molecular

markers: 12S rRNA (78.2 % of studies), with

COI, 16S rRNA, and Cytb collectively account-

ing for 20.2 %. The12S rRNA molecular mark-

er is commonly used for fish; the MiFish-U

primer set, designed specifically for teleosts by

Miya et al. (2015), accounts for 38 % of studies

in Takahashi et al. (2023), providing superior

detection and taxonomic resolution (Xu et al.,

2024) and relative abundance data (Shu et al.,

2021) in multi-primer tests).

Given these considerations, the purpose of

this pilot study was to use eDNA metabarcod-

ing targeting the 12S rRNA molecular marker

to obtain baseline data on the biodiversity

and composition of the fish species present in

the upstream and downstream reaches of the

Balantak River in Banggai Regency, Central

Sulawesi, Indonesia, one of many understudied

waterways in Sulawesi. Based on findings from

other Sulawesi rivers, the Balantak River was

expected to host predominantly diadromous

taxa including amphidromous Gobiiformes,

and to be at risk from non-native taxa. This

study provides baseline data for the Balantak

River that contributes to the body of knowledge

on the ichthyofaunal biodiversity in Sulawesi,

improving understanding of the native fish

fauna and providing information on the spread

of introduced species that are not native to

Sulawesi or the Wallacea region. These data

can inform conservation and fisheries manage-

ment, provide a reference point for research and

monitoring to understand the impact of human

activities on aquatic ecosystems, and pave the

way for improving and extending eDNA-based

approaches in Sulawesi and beyond.

MATERIALS AND METHODS

The Balantak River in Banggai Regency,

Central Sulawesi, Indonesia flows into the Gulf

of Tolo on the east coast of Sulawesi Island,

Indonesia and is typical of many such short riv-

ers originating in the mountainous hinterland

of the Banggai Regency. Two sampling sites

were selected (Fig. 1): Site 1 in the downstream

reaches (0°52’47” S & 123°22’37” E), and Site 2

in the upstream reaches of the Balantak River

(0°52’59” S & 123°23’18” E). These sites were

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

chosen to sample the ichthyofauna in the lower

reaches, flowing through the narrow coastal

plain close to the estuary, including euryhaline

taxa, as well as the ichthyofauna in the faster

flowing upper reaches of the Balantak River,

above the first rapids and waterfalls. Samples

were collected on 21 July 2024. Sterile surgi-

cal gloves were worn during sample collec-

tion, handling, and all laboratory procedures.

All equipment and work areas were sterilized

(10 % bleach and/or 96 % ethanol). Tweezers

and scissors were further sterilized using a

Bunsen burner immediately before use. Water

samples were collected in sterile plastic con-

tainers (approximately 5 L volume) and placed

in coolboxes filled with ice for transport to the

laboratory. All water samples were processed

following the Oceanogen protocol (Madduppa

& Sani, 2020). From each sample, 2 L of water

was filtered through a 0.45 µm Pall membrane

filter paper placed in a Nalgene rapid flow filter

attached to a one-stage vacuum pump (Allie

VE115K). The filters containing the DNA were

cut in half and each piece was placed in a 2 mL

cryo-tube (Greiner Bio-One GmbH) filled with

DNA/RNA Shield (Zymo Research). A negative

control was also obtained by filtering 1 L of

distilled water and treated in the same manner

to monitor potential contamination. The tubes

were labelled, sealed with parafilm and trans-

ported to Oceanogen in Bogor, Indonesia.

DNA extraction, amplification, and

sequencing were conducted at the Oceano-

gen Laboratory in Bogor, Indonesia. DNA was

extracted using Qiagen Blood and Tissue DNA

Extraction Kits following the manufacturer’s

instructions. The first PCR stage amplified the

target region of the 12S rRNA gene using the

MiFish-U primer set (Miya et al., 2015). The

12S rRNA marker and the MiFish-U primer set

were selected based on the considerations out-

lined above. The 12S rRNA marker has a com-

paratively high resolution for identifying teleost

fishes, in particular tropical freshwater taxa

and this relatively short DNA fragment is com-

patible with Next Generation metabarcoding

Fig. 1. Sampling sites on the Balantak River in Banggai Regency, Central Sulawesi, Indonesia.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

sequencing platforms. The MiFish-U primer

is widely used (Takahashi et al., 2023) and has

demonstrated comparatively high effectiveness

in amplifying teleost DNA across a wide range

of taxonomic groups (Xu et al., 2024; Zhu

et al., 2023). Furthermore, this primer set is

supported by a large and growing reference

sequence database and on-line data analysis

pipeline (Sato et al., 2018; Zhu et al., 2023). PCR

was performed on a 96 Universal peqStAR unit,

with three replicates per sample. PCR product

quality was visualized through electrophore-

sis. The three replicate PCR products from

each sample were then pooled, bead-cleaned

(AMPure XP) and indexed through a second

PCR stage with IDT double indices and Illu-

mina sequencing adapters for the Illumina

Nextera DNA Unique Dual Index Set B. The

indexed PCR product (amplicon library) was

purified using AMPure XP beads, quantified

using a Qubit fluorometer, and normalized to

equal concentrations prior to DNA sequencing

on a NovaSEQ6000.

Sequencing outputs (FASTQ files) were

analyzed using the MitoNGS option in the

MitoFish online portal (version 2025.06; Iwasa-

ki et al., 2013; Sato et al., 2018; Zhu et al., 2023).

Clean sequences were grouped into amplicon

sequence variants (ASVs) assigned to the clos-

est taxon in the GenBank 12S rRNA database

and produce denoised lists of taxa identified.

This process was repeated at two levels of

identity: 97 % ID (highly likely to be correct

at genus level) with minimum read abundance

0.01 and 99 % ID (likely to be accurate to spe-

cies level) without a minimum read abundance.

ASVs assigned to ray-finned fish (Actinopteri)

taxa were tabulated, analyzed and visualized

graphically in Microsoft Excel 365. The FASTQ

read files were submitted to the NCBI GenBank

Sequence Read Archive (SRA) under submis-

sion number SUB15073931, accession number

PRJNA1223125.

Taxonomic validity of assigned taxa was

verified through the World Register of Marine

Species (WoRMS Editorial Board, 2025) and

Eschmeyer’s Catalog of Fishes (Fricke et al.,

2025). Conservation status was obtained from

the (International Union for Conservation of

Nature [IUCN], 2025) Red List. Geographi-

cal distribution, residency status (endemic,

native, non-native) and human uses (food fish-

eries, ornamental fisheries, aquaculture) were

obtained from FishBase (Froese & Pauly, 2025)

and relevant literature. Indonesia spans three

major biogeographical regions (Sundaland,

Wallacea and the Sahul Shelf) with distinctive

freshwater ichthyofauna (Kottelat et al., 1993).

Therefore, the criterion for native residency sta-

tus was based on known native distributions in

Wallacea, and specifically in or near Sulawesi,

rather than in Indonesia. Ichthyofaunal com-

position at upstream and downstream sites was

described and compared graphically and with

reference to relevant literature.

RESULTS

The Actinopteri ASVs assigned by the

MitoFish pipeline in the “Species identified by

MitoFish” table at 97 and 99 % percent identity

thresholds were used for further analyses. ASVs

in the “Higher taxa identified by MitoFish”

table predominantly represented Gobiiformes

without close matches in the sequence database

and marine fish with low abundances. Non-fish

vertebrate ASVs (≈ 5 % of length-filtered reads

and 2-5 % of ASVs, excluded from further anal-

yses) were assigned to mammalian taxa from

four orders present in the study area and likely

to shed DNA in the river: Artiodactyla (Bos

sp., Sus sp.), Carnivora (Canis lupus), Primates

(Homo sapiens), Rodentia (Rattus sp.). The

number of reads and taxa assigned in the “Spe-

cies identified by MitoFish” table varied consid-

erably based on the percent identity threshold

(Table 1). At the 97 % confidence level 307

753 reads were assigned to 32 Actinopteri taxa

(putative species) from 12 orders, 15 families,

and 25 genera. At the 99 % confidence level

a total of 203 436 reads were assigned by the

MiFish pipeline to 19 Actinopteri taxa (puta-

tive species) from 10 orders, 12 families, and

17 genera.

Assigned read abundance and distribution

varied between the 12 orders assigned by the

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

MitoFish pipeline at the 97 % identity threshold

(Fig. 2A). Gobiiformes (42.66 %) were the most

abundant, closely followed by Anguilliformes

(39.91 %) and Centrarchiformes (14.94 %).

Cypriniformes read abundance was an order

of magnitude lower (1.46 %) and the remain-

ing seven orders (collectively 1.03 %) are not

distinguishable in Fig. 2A. Syngnathiformes

was only present downstream. Combined read

abundance upstream was lower for Centrarchi-

formes and higher for Gobiiformes and Anguil-

liformes compared to downstream.

Relative read abundance by family differed

between samples and sites, although similar

numbers of families were detected. The 15

families assigned at the 97 % identity threshold

(Fig. 2B) include three Gobiiformes families

(Gobiidae 26.13 %, Eleotridae 12.14 % and

Rhyacichthyidae 6.89 %) and two Centrarchi-

formes families (Kuhliidae and Terapontidae).

The remaining orders were each represented

by one family. Anguillidae had the highest

overall read abundance (39.91 %). Gobiiformes

and Centrarchiformes communities differed

between sites. Gobiidae reads were most abun-

dant at the upstream site, followed by Rhy-

acichthyidae, and Terapontidae. Eleotridae

were followed by Kuhliidae and Gobiidae at the

downstream station.

Agreement between the 97 and 99 % ID

lists at the genus level was high, with all taxa

identified at the 99 % level also detected at

the 97 % level. There were notable differences

between upstream and downstream commu-

nities, especially with respect to Gobiiformes

(Fig. 3). However, there were uncertainties

regarding some taxa at the species level. Several

ASVs were assigned to multiple species with ID

> 99 %, in particular anguillid eels and Gobi-

iformes (genera Sicyopterus and Glossogobius),

while some species-level assignments may rep-

resent closely related species lacking 12S rRNA

reference sequences in the GenBank database.

The taxa assigned to the 32 ASVs in the

“Species identified by MitoFish” table at the

97 % identity threshold, including alternative

Table 1

Summary of reads, ASVs and taxa assigned by the MiFish pipeline at 97 and 99 % sequence percentage identity thresholds

for eDNA samples from two sites on the Balantak River in Banggai Regency, Central Sulawesi.

Parameter/ID level Site 1 Site 2

Downstream 1 Downstream 2 Upstream 1 Upstream 2

Total reads-raw 507 834 556 680 625 460 536 414

Assembled reads after quality-filter, no N 359 529 401 556 415 409 341 175

Reads after length filter 147 752 154 683 209 391 194 456

Unique reads 13 973 14 157 15 587 14 051

Denoised ASVs 207 304 146 90

Actinopteri ASVs 202 300 142 86

Number of Actinopterygii Orders at species-level

ID level 97 % 12 11 11 10

ID level 99 % 8 8 9 8

Number of Actinopteri Families

ID level 97 % 14 14 14 13

ID level 99 % 10 10 11 9

Number of Actinopteri Genera

ID level 97 % 19 23 24 18

ID level 99 % 12 14 16 10

Number of Actinopteri species level taxa/species identified without ambiguity

ID level 97 % 20/15 25/19 30/22 22/17

ID level 99 % 11/10 17/13 18/13 11/8

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

identifications where given, included species

assessed as at risk of extinction in the IUCN

Red List (IUCN, 2025); endemic, native, and

non-native taxa, and taxa of uncertain status;

and fish caught or cultured as food and/or for

ornamental use (Table 2). Among the identi-

fied taxa, Sicyopterus (Gobiidae) exhibited the

greatest species diversity.

Fig. 2. Actinopteri orders (A) and families (B) detected through eDNA metabarcoding at downstream and upstream

sampling sites on the Balantak River in Banggai Regency, Central Sulawesi, Indonesia (MitoFish, 97 % identity threshold).

Table 2

Conservation status, origin, use, and previous reports from Sulawesi of taxa (ASVs) detected through eDNA barcoding at

two stations in the Balantak River, Banggai Regency, Central Sulawesi, Indonesia (MitoFish 97 % identity threshold, 0.01 %

read abundance threshold).

No Order Family Genus/Species StatusaOriginbUsecReportc

1Anabantiformes Channidae Channa striata LC U A,F Yes

2 Anguilliformes Anguillidae Anguilla interioris DD N F Yes

A. marmorata LC N F Yes

3Atheriniformes Telmatherinidae Telmatherina celebensis NT E F,O Ye s d

4Beloniformes Adrianichthyidae Oryzias celebensis LC E O Ye s e

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No Order Family Genus/Species StatusaOriginbUsecReportc

5Centrarchiformes Kuhliidae Kuhlia marginata LC N F Ye s f

6Centrarchiformes Terapontidae Leiopotherapon plumbeus

L. unicolor

7 Cichliformes Cichlidae Oreochromis niloticus LC A A,F Ye s g

8 Cichliformes Cichlidae Cichlidae sp. - A O Ye s g

9 Cypriniformes Cyprinidae Osteochilus sp. CBM-ZF-12 420 - A F Ye s i

10 Gobiiformes Eleotridae Belobranchus belobranchus LC NO,FjYe s

11 Gobiiformes Eleotridae Eleotris fusca LC NO,FjYe s

12 Gobiiformes Eleotridae Giuris tolsoni NE N F Ye s n

13 Gobiiformes Gobiidae Glossogobius illimis LC N - Ye s

14 Gobiiformes Gobiidae G. aureus

G. flavipinnis

FYes

Ye s d

15 Gobiiformes Gobiidae Lentipes armatus NE U OkNo

16 Gobiiformes Gobiidae L. palawanirufus NE U OkNo

17 Gobiiformes Gobiidae Schismatogobius sp. CBM:ZF:12 245 - - Fj-

18 Gobiiformes Gobiidae Sicydium altum VU A - No

19 Gobiiformes Gobiidae Sicyopterus aiensis

S. cynocephalus

S. lengguru

F,O

Yes

20 Gobiiformes Gobiidae S. lagocephalus LC N Ok,FjYe s j

21 Gobiiformes Gobiidae S. longifilis LC N Ok,FjYe s j

22 Gobiiformes Gobiidae S. marquesensis DD U F No

23 Gobiiformes Gobiidae S. squamosissimus NE U OkNol

24 Gobiiformes Gobiidae S. longifilis

S. squamosissimus

See above

25 Gobiiformes Gobiidae Sicyopus zosterophorus LC N Ok,FjYes

26 Gobiiformes Gobiidae Smilosicyopus leprurus LC N Ok,FjYes

27 Gobiiformes Gobiidae Stiphodon semoni LC N Ok,FjYe s

28 Gobiiformes Rhyacichthyidae Rhyacichthys aspro DD N Ok,F Yes

29 Gonorynchiformes Chanidae Chanos chanos LC N A,F Ye s

30 Mugiliformes Mugilidae Planiliza subviridis

Mugil cephalus

Yes

Ye s m

31 Siluriformes Clariidae Clarias batrachus LC A A,F,O Ye s g

32 Syngnathiformes Syngnathidae Microphis (formerly Coelonotus)

leiaspis

LC N OkYes

Table caption: a IUCN Red List assessments (IUCN, 2025): LC = Least Concern; DD = Data Deficient; EN = Endangered;

NT = Near Threatened; NE = not evaluated; - not assigned to species level. b IUCN Red List (IUCN, 2025) and FishBase

(Froese & Pauly, 2025): A = alien, reported native distribution does not include Sulawesi; E = Sulawesi endemic; N =

native; U = unclear; - no data. c IUCN Red List (IUCN, 2025) and FishBase (Froese & Pauly, 2025) unless indicated by

further superscripts: A = aquaculture; F = food; O = ornamental; - = no data; Report = reported from Sulawesi. d Currently

considered endemic to the Malili Lake complex (Lumbantobing, 2019b; Lumbantobing & Larson, 2019). e Reported from

South and Southeast Sulawesi and a site in Timor Leste, but not from watersheds close to the study area. f (Haryono & Sauri,

2020; Miesen et al., 2016; Tweedley et al., 2013; Wantania et al., 2024). g Many reports of these and other Cichliformes in

Sulawesi waters (see discussion). h (Astuti et al., 2023; Herder et al., 2022; Wantania et al., 2024). i Reports at genus level

(Herder et al., 2022; Herjayanto et al., 2019; Ndobe et al., 2024; Serdiati et al., 2020; Serdiati et al., 2024; Wantania et al.,

2024). j Amphidromous species fished for food as post-larvae, locally called nike or penja in Sulawesi waters (Nurjirana et

al., 2019; Nurjirana et al., 2021; Nurjirana et al., 2022; Pangemanan et al., 2020; Sahami et al., 2020). k Traded on-line in the

ornamental fish trade (advertisements/hobbyist posts present on 30 Nov 2024). l recently described from Java and Sumatra

(Keith et al., 2015). m Currently considered a species complex with at least 15 species and likely confusion with several other

genera in Mugilidae (Durand et al., 2017). n Reported from several sites in Sulawesi, including Luwuk (Keith et al., 2020).

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Fig. 3. Heatmap of Actinopteri genera (A), fill color from blue to yellow as assigned read volume increases; and relative (read)

abundance of Gobiiformes genera (B) detected through eDNA metabarcoding at downstream and upstream sampling sites

on the Balantak River in Banggai Regency, Central Sulawesi, Indonesia (MitoFish, 97 % identity thresholds).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

DISCUSSION

The ability to detect species through eDNA

not only enhances conservation efforts but also

facilitates the management of aquatic resources

by providing critical data on species distribu-

tion and abundance. At the study sites on

the Balantak River in Banggai Regency, Cen-

tral Sulawesi, eDNA metabarcoding identi-

fied Actinopteri (ray-finned fish) amplicon

sequence variants (ASVs) representing at least

32 species-level taxa. of which 19 belong to the

Gobiiformes. The observed conditions, includ-

ing clear, fast-flowing waters and lush riparian

vegetation, with little evidence of direct habitat

degradation, provide habitat suitable for many

amphidromous Gobiiformes (Keith & Lord,

2012; Keith, Lord et al., 2014), as evidenced by

the high proportion (59 %) of gobies among the

taxa identified. While these results do not imply

that the Balantak River is free from threats to

aquatic biodiversity, they indicate that this river

supports a rich biodiversity worth conserving.

The ichthyofaunal community composi-

tion differed between the upstream and down-

stream stations, including endemic, native and

alien species, as well as taxa of uncertain ori-

gin. The most abundant non-anguillid ASVs

at the downstream station included Eleotris

fusca Bloch and Schneider 1801, Kuhlia mar-

ginata Cuvier 1829, Giuris tolsoni Bleeker 1854,

Leiopotherapon sp., Glossogobius illimis Hoese

and Allen 2012, and Sicyopterus longifilis de

Beaufort 1912. At the upstream station, the

most abundant non-anguillid ASVs were all

Gobiiformes, including Smilosicyopus leprurus

Sakai and Nakamura 1979, Rhyacichthys aspro

Valenciennes 1837, Sicyopterus sp. (likely S.

cynocephalus Valenciennes 1837), Belobranchus

belobranchus Valenciennes 1837, Sicyopus zos-

terophorus Bleeker 1856, Lentipes armatus Sakai

& Nakamura 1979 and L. palawanirufus Maeda

and Palla 2021.

The majority of ASVs (24, 75 %) were

assigned to euryhaline or diadromous taxa

that spend some stages of their life cycles in

marine waters. The most numerous were the

amphidromous Gobiiformes (20 ASVs), while

the sole Syngnathiformes taxon, the barhead

pipefish Microphis leiaspis Bleeker, 1854 (for-

merly Coelonotus leiaspis), is also amphidro-

mous (Jenkins et al., 2017). This pipefish was

only identified at the downstream site, likely

due to topography. This is consonant with a

longitudinal survey of the ichthyofauna in the

nearby Koyoan River (Ndobe et al., in press),

also in Banggai Regency, where M. leiaspis was

only observed in the lower reaches of the river,

below the first waterfall. Together, these studies

indicate that, despite the ability to swim past

some rapids (Ishihara & Tachihara, 2008), this

pipefish is likely unable to pass waterfalls to

reach upstream montane habitat in these short,

steep rivers.

The predominance of Gobiiformes taxa is

not surprising, given that this order is the most

speciose in Sulawesi and other Wallacean fresh-

waters (Kottelat et al., 1993; Kottelat & Whitten,

1996). The three catadromous taxa assigned

are all widespread species likely present in the

study area, namely anguillid eels (Pike et al.,

2020); the greenback mullet Planiliza subviridis

Valenciennes, 1836 (Bru, 2022); and the silver

flagtail Kuhlia marginata (Hoese et al., 2019)

reported from rivers in the Banggai Islands,

close to the study site (Haryono & Sauri, 2020).

The milkfish Chanos chanos Forsskål, 1 775

is a widespread primarily marine euryhaline

species known to enter estuaries and rivers

and is popular in brackish-water aquaculture

(Freyhof et al., 2019).

Multiple anguillid ASVs (one to five per

station) had 99.5 %-100 % similarity to both

the highlands long-finned eel Anguilla inte-

rioris Whitley 1938 and the giant mottled eel

Anguilla marmorata Quoy & Gaimard 1824.

Both A. interioris and A. marmorata have been

reported from the surrounding region, and

sympatric occurrence of anguillid eels is com-

mon in Sulawesi rivers and lakes (Aoyama

et al., 2018; Arai, 2016; Hagihara et al., 2018;

Serdiati et al., 2023; Sugeha, 2010). Both taxa

may well be present, although the high simi-

larities (> 99.5 %) with reference sequences for

both species indicate that the 12S rRNA mark-

er used cannot reliably distinguish between

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

these two congeners. This indication was

reinforced by a phylogenetic reconstruction

using ASV sequences from this study and all

available 12SrRNA reference sequences for the

genus Anguilla in the NCBI GenBank data-

base (unpublished, available from the authors),

where accessions of the two species had up to

100 % identity. The presence of anguillid eels

is important from fisheries and conservation

perspectives, as eels are valuable food fish

and play important though still poorly under-

stood ecological roles in tropical watersheds

(Arai, 2016; Drouineau et al., 2018; Righton

et al., 2021; Tembo et al., 2023). Tropical eels

are heavily exploited from glass eel to silver

eel stages globally (Jacoby et al., 2015) and in

Sulawesi (Ambo-Rappe & Moore, 2019), often

likely exceeding sustainable limits (Righton et

al., 2021). Studies on anguillid eels cover many

other regions of Sulawesi, including Poso Lake,

Poso River and Tomini Bay; North Sulawesi and

the Sulawesi Sea; the Makassar Strait including

Palu Bay and the East coast of Sulawesi (Arai,

2016; Arai et al., 1999; Serdiati et al., 2023;

Sugeha, 2010; Triyanto et al., 2021). However,

although A. marmorata is reported from Peleng

Island in the Banggai Archipelago, separated

from the study area by a strait 30-50 km wide

(Haryono & Sauri, 2020), data are lacking on

anguillid eel populations and fisheries along

the East coast of Sulawesi facing the Gulf of

Tolo and Banda Sea. This study indicates that

this lack of data does not reflect a lack of eels,

highlighting the need for research on the genus

Anguilla in this region.

The identification of terapontid Centrar-

chiformes ASVs as Leiopotherapon plumbeus

Kner 1864 or L. unicolor Günther 1859 is like-

ly a case of misidentification. Leiopotherapon

plumbeus is considered endemic to the Philip-

pines (Torres et al., 2021), while L. unicolor is

considered restricted to Australia (Kennard

et al., 2019). The only freshwater terapontid

reported from Sulawesi is Lagusia micracanthus

Bleeker, 1860, endemic to the Maros region

of South Sulawesi (Hidayani et al., 2024; Vari

& Hadiaty, 2012). Until recently, this species

lacked COI DNA barcode sequences (Hidayani

et al., 2024) and still lacks 12S rRNA refer-

ence sequences. The comparatively low identity

(97-98 % ID) indicates that this detection is

more likely to represent a range extension for a

species lacking 12S rRNA references sequenc-

es (e.g., L. micracanthus) or an undescribed

terapontid rather than a range extension for L.

plumbeus or L. unicolor.

Primary freshwater fish taxa detected

included two Sulawesi endemic species: Tel-

matherina celebensis Boulenger, 1897 and Ory-

zias celebensis Weber, 1894. The sailfin silverside

T. celebensis is considered endemic to the Malili

ancient lake complex (Lumbantobing, 2019b)

and is more likely a misidentification than a

range extension of this species. Reasons for

erroneous assignment include limitations of

the molecular marker used in distinguishing

between species in some taxa (Porter & Hajiba-

baei, 2018), such as Telmatherinidae (Hubert

et al., 2015), and database gaps due to limited

research on riverine Telmatherinidae (Stelbrink

et al., 2014), especially in Eastern Sulawesi. The

Celebes ricefish Oryzias celebensis (Lumban-

tobing, 2019a) is relatively widespread, and if

confirmed this detection will extend its known

range within Sulawesi. Likely introduced dur-

ing prehistoric times, with a long history in

culinary culture and considered native by local

people, the origin of striped snakehead Channa

striata Bloch, 1793 populations in Sulawesi

remains uncertain (Ndobe et al., 2023).

Four alien invasive species detected in this

study have been reported from Sulawesi in con-

nection with deliberate introductions or releases

from aquaculture (Herder et al., 2022; Herjay-

anto et al., 2019; Serdiati et al., 2020; Yanuarita

et al., 2020). Barbs Osteochilus sp. and catfish

Clarias batrachus Linnaeus 1758 are native to

areas in Sundaland but not Sulawesi (Astuti et

al., 2023; Chua & Lim, 2019; Ng & Low, 2019;

Thinh et al., 2020). Barbs have been released

in some Sulawesi freshwater bodies to increase

fisheries yield (Yanuarita et al., 2020); alongside

tilapia and other cichlids, they are among the

many alien species potentially implicated in the

decline of native fish in Sulawesi lakes (Herder

et al., 2022; Serdiati et al., 2020). Tilapia in

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Indonesia originated from Africa (Robin et al.,

2023). In addition to O. niloticus, Oreochromis

ASV’s not identified to species level (< 97 %

identity) may indicate the presence of hybrids

or strains common in Indonesian aquaculture

as reported from other Indonesian freshwater

bodies (Herder et al., 2022; Ndobe et al., 2023;

Yanuarita et al., 2020). The taxon assigned as

Cichlidae sp. may be a hybrid. Cichlids released

in Indonesia waters are primarily of South

American or African origin (Robin et al., 2023)

and at least one hybrid cichlid (dubbed flow-

erhorn cichlid) has been reported from other

Sulawesi freshwaters, including the Malili lake

complex (Hilgers et al., 2018).

The detection of alien species should be

considered a wake-up call to evaluate and

implement measures to prevent, eradicate, or

manage the spread of invasive species that may

negatively impact native biodiversity (Sorensen,

2021). On a recent map showing global hotspots

of established alien species, most of Indonesia

falls within the top 50 % category, and Sulawesi

Island in the top 10 % category (Pyšek et al.,

2020). Introduced species can cause harm to

native species, populations, communities, and

ecosystems through direct and indirect routes

or processes, including predation, competition,

hybridization, disease transmission, altering

food-webs and energy flows (Bernery et al.,

2022; Dudgeon, 2020; Sorensen, 2021). World-

wide and across taxonomic groups, there is

strong evidence that alien species invasions

should be monitored and either eradicated or

controlled, as interventions are more effective

and less costly when detected and addressed

at an early stage (Bernery et al., 2022; IPBES,

2023; Pyšek et al., 2020; Sorensen, 2021). Even

when eradication of well-established invasive

taxa is not feasible, impact mitigation remains

an important strategy (Bernery et al., 2022;

IPBES, 2023). Read abundances for invasive

alien species detected in the Balantak River

were comparatively low (1.55 %) but occurred

at both stations. While these detections could

represent the release of DNA from nearby

fish-farms or waste from fish purchased and

consumed by people living nearby (rather than

from wild populations of these species), this

is unlikely at the upstream site, as aquaculture

and human habitation are concentrated in the

coastal plain. The presence of non-native fish

DNA, especially at the upstream site, could

signal early stages of alien species invasion

processes. If the presence of potentially invasive

fish species is confirmed, management mea-

sures for eradication or control might be fea-

sible and appropriate (Dudgeon, 2020; IPBES,

2023; Pyšek et al., 2020; Sorensen, 2021).

Most native Gobiiformes taxa identified in

this study have been reported from other rivers

in Banggai Regency, in particular B. belobran-

chus, E. fusca, Sicyopterus lagocephalus Pallas

1770, Sicyopterus longifilis, Stiphodon semoni

Weber 1895, and Sicyopus zosterophorus (Gani

et al., 2019; Gani, Adam et al., 2021). These

studies also found specimens assigned to genus

level but not identified to species level, espe-

cially in the genera Lentipes, Schismatogobius

and Sicyopterus. A phylogenetic reconstruction

(not included in this study) for the genus Sicyo-

pterus indicated eight likely species-level taxa,

with two unambiguously identified at the 99

% level (S. lagocephalus, S. longifilis). It is also

worth noting that Gobiiformes taxonomy is

under revision and could give rise to confusion;

for example, the taxon E. fusca is now thought

to comprise several cryptic species (Mennesson

et al., 2018). Furthermore, a large proportion

of Actinopteri reads were assigned to Gobi-

idae with a high degree of confidence but not

assigned at lower taxonomic levels.

Assigned taxa from the Gobiidae, Tera-

pontidae, and Mugilidae with reported dis-

tributions that do not include Sulawesi (Table

2) are unlikely to be introduced alien species.

Some could be misidentifications due to data-

base gaps or erroneous sequences. However,

based on the distributions of other sicydiine

gobies (Keith, Lord et al., 2014), new species

discoveries and recent range extensions (e.g.,

an amphidromous goby previously considered

restricted to the Philippines recently found in

Sulawesi (Ndobe et al., 2023), some assign-

ments may represent range extensions. L. pala-

wanirufus was described from the Philippines

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

(Maeda et al., 2021) while L. armatus Sakai

& Nakamura, 1979, previously considered

restricted to Japan and Taiwan (Keith, Hadiaty

et al., 2014) has since been reported from the

Philippines (Tauli et al., 2022). One or both

may well be present in Sulawesi, and if con-

firmed would represent similar range exten-

sions to the recent records for nearby rivers of

Stiphodon annieae Keith, Hadiaty et al. (2014),

(Gani, Nurjirana et al., 2021) and S. surrufus

Watson & Kottelat 1995 (Ndobe et al., 2022).

Four Sicyopterus taxa given as alternative or

sole identifications by the MitoFish pipeline are

not recorded from areas close to the study site:

S. aiensis Keith, Watson and Marquet (2004)

is considered endemic to Vanuatu (Lord et

al., 2010), S. marquesensis Fowler, 1932 to the

Marquesas Islands in French Polynesia (Boseto,

2015); S. lengguru Keith, Lord et al. (2012) was

described from Papua New Guinea (Keith et

al., 2011); and S. squamosissimus from Java and

Bali, Indonesia (Keith et al., 2015).

In addition to taxonomic uncertainties,

the taxa identified in this study do not neces-

sarily reflect the full fish diversity present in

the Balantak River. The eDNA approach used

relies on DNA traces shed by organisms pres-

ent at or near the sampling stations and then

collected in relatively small water samples, and

there are many reasons why DNA from taxa

present in the environment could be missed or

could fail to be detected (Porter & Hajibabaei,

2018; Ruppert et al., 2019; Shu et al., 2021). As

identification tools, molecular methods such

as DNA barcoding and eDNA metabarcoding

rely on the presence of homologous reference

sequences; gaps or errors in the databases used

can lead to misidentification or non-identi-

fication (McElroy et al., 2020; Moore et al.,

2021; Moore et al., 2025; Porter & Hajibabaei,

2018). Other challenges in eDNA metabarcod-

ing include the risk of contamination (false

positives), so-called primer bias (likelihood of

amplification across taxonomic groups), com-

plex laboratory and bioinformatic analyses,

technical (e.g. nucleotide read) errors, and

interpretation of results (Porter & Hajibabaei,

2018; Shu et al., 2021; Wang et al., 2024). In this

study, negative samples were not collected in

the field, but laboratory negative controls were

negative. Discrepancies can arise due to DNA

degradation before or after collection, as deg-

radation rates are influenced by factors such as

UV exposure, temperature, microbial activity,

and pH levels (Perry et al., 2024; Ruppert et al.,

2019). Reduced DNA quality from individuals

presents some time ago may make detection

less likely and increase the likelihood of errone-

ous read sequences (Porter & Hajibabaei, 2018).

We could not find directly relevant data on

DNA degradation for the methods, conditions

and taxa in this study, but it is a possible source

of unassigned ASVs and could have resulted in

false negatives.

Even if DNA from all fish present was

detected and correctly identified, the results

are intrinsically limited as fish can move ran-

domly or following patterns at various spatial

and temporal scales (Perry et al., 2024), and

riverine fish communities can change over

time, for example between seasons (Wantania

et al., 2024). Furthermore, some fish (e.g.,

large-bodied species or adult individuals) may

shed more eDNA than others (e.g., smaller fish

or juvenile stages) (Maruyama et al., 2014),

and some taxa are more easily amplified and

detected than others during the laboratory pro-

cesses (Miya et al., 2020). Despite the possibility

of false positives due to fish DNA from food

waste at the downstream site, the taxa identified

likely underestimate the ichthyofaunal diversity

of the Balantak River. This is indicated by the

high proportion of Actinopteri reads assigned

to family level (predominantly Gobiidae) with

a high degree of confidence but not assigned

at lower taxonomic levels. This study was con-

ducted during the South or East monsoon. Fur-

ther sampling to account for seasonal variation

would provide a better picture of ichthyofaunal

composition. Furthermore, given the large pro-

portion of diadromous species, sampling over

a greater spatial as well as temporal span (e.g.,

across watersheds in the region) would enable a

better assessment of species status as well as fish

biodiversity in the many short rivers along the

East coast of the Banggai Regency.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

This study contributes baseline data for

natural resources in Central Sulawesi, at the

heart of the Wallacea bioregion. The results call

for further research, in particular integrative

taxonomic studies to validate or correct some

of the taxonomic assignments, and fill gaps in

public sequence databases (Haÿ et al., 2023;

Riley et al., 2023). Once the presence of a spe-

cies is confirmed, its local conservation status

can be evaluated, and if necessary, management

strategies (in-situ and, if necessary ex-situ) can

be developed and implemented (Maasri et al.,

2022). All native genera identified are known

to be exploited for human use (Table 1), and the

gaps in IUCN Red List coverage highlight the

ongoing need for assessments. The majority of

taxa detected were diadromous or euryhaline,

reinforcing the importance of maintaining con-

nectivity between coastal and riverine ecosys-

tems and throughout watersheds (Shao et al.,

2019; Lagarde et al., 2021; Wantania et al., 2024).

This study demonstrates that many ecologically

and economically important species are using

both marine and freshwater environments in

this and nearby watersheds during their life-

cycles. Therefore, it is vital to protect both

environments, and the land-sea interface where

they meet, crossing institutional and adminis-

trative boundaries in terms of governance and

stakeholder groups with an interdisciplinary

and collaborative approach. Furthermore, the

detection and control of non-native species and

the prevention of further introductions should

be taken seriously in the Balantak River, and

across all Sulawesi freshwaters.

While long-term conservation of fresh-

water ecosystems, their biodiversity, and

ecosystem services require addressing major

global challenges, especially related to climate

change, local action is vital (Dudgeon, 2019;

Oikonomou, 2021). Management strategies for

Sulawesi freshwaters, such as the Balantak River

watershed, should focus on conserving the rich

native ichthyofauna, in particular diadromous

species, while eliminating or controlling alien

invasive species. Options include but are not

limited to habitat protection or management;

fisheries management including data collection

and regulations, especially for ornamental fish-

eries and trade; conservation breeding and

sustainable aquaculture practices that provide

both conservation and economic benefits; and

developing a comprehensive and reliable 12S

rRNA database for Sulawesi freshwater fishes

supported by vouchered specimens.

Author contribution statement: Con-

ception and design of the study: all authors;

Investigation: Magdalena Litaay, Widyastuti

Umar, Abdul Gani, Arfiani Rizki Paramata;

Validation: Magdalena Litaay, Asmi Citra Mali-

na Tassakka, Nadiarti Nurdin Kadir, Samliok

Ndobe; Data analysis: Nadiarti Nurdin Kadir,

Widyastuti Umar, Arfiani Rizki Paramata, Abi-

gail Mary Moore; Interpretation of results: all

authors; Writing – original draft: Magdalena

Litaay, Abigail Mary Moore; Writing - review

and editing: all authors. Funding acquisition:

Magdalena Litaay.

Ethics statement: The authors declare that

they all agree with this publication and that

they have made contributions that justify their

authorship; that there is no conflict of interest

of any kind; and that they have complied with

all relevant ethical and legal requirements and

procedures. All sources of funding are fully and

clearly detailed in the acknowledgements sec-

tion. The respective signed legal document is in

the journal’s archives.

ACKNOWLEDGMENTS

The authors gratefully acknowledge sup-

port from the Hasanuddin University 2024

Research Grant scheme with contract number:

00309/UN4.22/PT.01.03/2024. We also thank

Suharto for assistance with the site map and

all students and colleagues who assisted in the

field and the laboratory.

REFERENCES

Albert, J. S., Destouni, G., Duke-Sylvester, S. M., Magurran,

A. E., Oberdorff, T., Reis, R. E., Winemiller, K. O., &

Ripple, W. J. (2021). Scientists’ warning to humanity

16 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

on the freshwater biodiversity crisis. Ambio, 50(1),

85–94. https://doi.org/10.1007/s13280-020-01318-8

Ambo-Rappe, R., & Moore, A. M. (2019). Sulawesi Seas,

Indonesia. In C. Sheppard (Ed.), World Seas: An Envi-

ronmental Evaluation (2nd ed., pp. 559–581). Elsevier.

https://doi.org/10.1016/B978-0-08-100853-9.00032-4

Aoyama, J., Wouthuyzen, S., Miller, M. J., Sugeha, H. Y.,

Kuroki, M., Watanabe, S., Syahailatua, A., Tantu, F.

Y., Hagihara, S., Triyanto, T., Otake, T., & Tsukamoto,

K. (2018). Reproductive ecology and biodiversity of

freshwater eels around Sulawesi Island Indonesia.

Zoological Studies, 57, 30. https://doi.org/10.6620/

ZS.2018.57-30

Arai, T., Aoyama, J., Limbong, D., & Tsukamoto, K. (1999).

Species composition and inshore migration of the

tropical eels Anguilla spp. recruiting to the estuary

of the Poigar River, Sulawesi Island. Marine Ecology

Progress Series, 188, 299–303. https://doi.org/10.3354/

meps188299

Arai, T. (2016). Biology and ecology of anguillid eels. CRC

Press.

Astuti, S. S., Hariati, A. M., Kusuma, W. E., Yuniarti,

A., Kurniawan, N., & Wiadnya, D. G. R. (2023).

Anthropogenic introduction of the spotted barb, Bar-

bodes binotatus, across the Wallace Line in western

Sulawesi, Indonesia. Biodiversitas, 24(3), 1916–1925.

https://doi.org/10.13057/biodiv/d240369

Bartholomew, D. C., Mosyaftiani, A., Morgan, B., Shah, T.,

Shaw, K., Stillman, C., Baldwin, K., Baqueiro, L. H.

R., Birkinshaw, C., Breman, E., Diniz, G., Fereira, P.,

Flowers, C., Gichira, A., Jenkins, J., Kindt, R., Molin,

P. G., Mungai, T., & Gann, G. D. (2024). The global

biodiversity standard: Manual for assessment and best

practices. Botanic Gardens Conservation Internatio-

nal & Society for Ecological Restoration.

Bernery, C., Bellard, C., Courchamp, F., Brosse, S.,

Gozlan, R. E., Jarić, I., Teletchea, F., & Leroy, B.

(2022). Freshwater fish invasions: A comprehensive

review. Annual Review of Ecology, Evolution, and

Systematics, 53(1), 427–456. https://doi.org/10.1146/

annurev-ecolsys-032522-015551

Boseto, D. (2015). Sicyopterus marquesensis. The IUCN Red

List of Threatened Species 2015: e.T196365A2449948.

https://doi.org/10.2305/IUCN.UK.2012.RLTS.

T196365A2449948.en

Bru, E. (2022). Planiliza subviridis, greenback mullet.

The IUCN Red List of Threatened Species. https://

doi.org/10.2305/IUCN.UK.2022-1.RLTS.

T197050A2478575.en

Chen, X., Li, S., Zhao, J., & Yao, M. (2024). Passive eDNA

sampling facilitates biodiversity monitoring and rare

species detection. Environment International, 187,

108706. https://doi.org/10.1016/j.envint.2024.108706

Chua, K. W. J., & Lim, K. (2019). Barbodes binotatus,

common barb. The IUCN Red List of Threatened Spe-

cies. https://doi.org/10.2305/IUCN.UK.2019-2.RLTS.

T169538A90996154.en

Drouineau, H., Durif, C., Castonguay, M., Mateo, M.,

Rochard, E., Verreault, G., Yokouchi, K., & Lambert,

P. (2018). Freshwater eels: A symbol of the effects

of global change. Fish and Fisheries, 19(5), 903–930.

https://doi.org/10.1111/faf.12300

Dudgeon, D. (2019). Multiple threats imperil freshwa-

ter biodiversity in the Anthropocene. Current Bio-

logy, 29(19), R960–R967. https://doi.org/10.1016/j.

cub.2019.08.002

Dudgeon, D. (2020). Freshwater biodiversity. Cambridge Uni-

versity Press. https://doi.org/10.1017/9781139032759

Durand, J. D., Hubert, N., Shen, K.-N., & Borsa, P. (2017).

DNA barcoding grey mullets. Reviews in Fish Biology

and Fisheries, 27(1), 233–243. https://doi.org/10.1007/

s11160-016-9457-7

Freyhof, J., Sparks, J. S., Kaymaram, F., Feary, D., Bishop, J.,

Al-Husaini, M., Almukhtar, M., Hartmann, S., Alam,

S., & Al-Khalaf, K. (2019). Chanos chanos, milkfish

(amended version). The IUCN Red List of Threatened

Species. https://doi.org/10.2305/IUCN.UK.2019-2.

RLTS.T60324A151598011.en

Fricke, R., Eschmeyer, W. N., & Van der Laan, R. (Eds.).

(2025). Eschmeyer’s catalog of fishes: Genera, spe-

cies, references, electronic version. California Academy

of Sciences. http://researcharchive.calacademy.org/

research/ichthyology/catalog/fishcatmain.asp

Froese, R., & Pauly, D. (Eds.). (2025). FishBase: The global

database of fishes. https://www.fishbase.se/

Gani, A., Bakri, A. A., Adriany, D. T., Nurjirana, N., Herja-

yanto, M., Bungalim, M. I., Ndobe, S., & Burhanuddin,

A. I. (2019). Identification of freshwater goby species

from the Biak and Koyoan Rivers, Luwuk Banggai,

Central Sulawesi. Jurnal Ilmu Kelautan SPERMONDE,

5(2), 57–60. https://doi.org/10.20956/jiks.v5i2.8931

Gani, A., Nurjirana, N., Bakri, A. A., Adriany, D. T.,

Wuniarto, E., Khartiono, L. D., Satria, D. H., Hasan,

V., Herjayanto, M., Burhanuddin, A. I., Moore, A.

M., & Kobayashi, H. (2021). First record of Stiphodon

annieae Keith & Hadiaty, 2015 (Teleostei, Oxuder-

cidae) from Sulawesi Island, Indonesia. Check List,

17(1), 261–267. https://doi.org/10.15560/17.1.261

Gani, A., Adam, M. I., Bakri, A. A., Adriany, D. T., Herja-

yanto, M., Nurjirana, Mangitung, S. F., & Andriyono,

S. (2021). Diversity studies of freshwater goby species

from three river ecosystems in Luwuk Banggai, Cen-

tral Sulawesi, Indonesia. IOP Conference Series: Earth

and Environmental Science, 718(1), 012087. https://

doi.org/10.1088/1755-1315/718/1/012087

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Hagihara, S., Aoyama, J., Limbong, D., & Tsukamoto, K.

(2018). Interspecific and sexual differences in riverine

distribution of tropical eels Anguilla spp. Journal of

Fish Biology, 93(1), 21–29. https://doi.org/10.1111/

jfb.13666

Hansen, H. H., Bergman, E., Cowx, I. G., Lind, L., Pauna, V.

H., & Willis, K. A. (2023). Resilient rivers and connec-

ted marine systems: A review of mutual sustainability

opportunities. Global Sustainability, 6, e2. https://doi.

org/10.1017/sus.2022.19

Haryono, H., & Sauri, S. (2020). Komunitas ikan di perai-

ran tawar wilayah Kabupaten Banggai Kepulauan

Sulawesi Tengah. Sriwijaya Bioscientia, 1(1), 1–7.

https://doi.org/10.24233/sribios.1.1.2020.164

Haÿ, V., Mennesson, M. I., Lord, C., & Keith, P. (2023).

Why several when one can unite them all? Integrative

taxonomic revision of Indo-Pacific freshwater pipe-

fish (Nerophinae). Zoological Journal of the Linnean

Society, 198(4), 923–956. https://doi.org/10.1093/

zoolinnean/zlad007

Herder, F., Schliewen, U., Geiger, M., Hadiaty, R., Gray, S.,

McKinnon, J., Walter, R., & Pfaender, J. (2012). Alien

invasion in Wallace’s Dreamponds: Records of the

hybridogenic “flowerhorn” cichlid in Lake Matano,

with an annotated checklist of fish species introdu-

ced to the Malili Lakes system in Sulawesi. Aqua-

tic Invasions, 7(4), 521–535. https://doi.org/10.3391/

ai.2012.7.4.009

Herder, F., Möhring, J., Flury, J. M., Utama, V., Wantania,

L., Wowor, D., Boneka, F. B., Stelbrink, B., Hilgers,

L., Schwarzer, J., & Pfaender, J. (2022). More non-

native fish species than natives, and an invasion of

Malawi cichlids, in ancient Lake Poso, Sulawesi,

Indonesia. Aquatic Invasions, 17(1), 72–91. https://

doi.org/10.3391/ai.2022.17.1.05

Herjayanto, M., Gani, A., Adel, Y. S., & Suhendra, N. (2019).

Freshwater fish of lakes and its inlet rivers in Sulawesi

Tengah Province, Indonesia. Journal of Aquatropica

Asia, 4(1), 1–9. https://doi.org/10.33019/aquatropica.

v4i1.1679

Hidayani, A. A., Tassaka, A. C. M. A. R., Kadir, N. N., Yasir,

I., Jompa, J., Umar, W., Litaay, M., Parenrengi, A.,

Ndobe, S., Gani, A., Putri, A. P., Iqram, M., Omar, S.

B. A., & Moore, A. M. (2024). First DNA barcodes

of two endemic fishes from the Maros karst region,

South Sulawesi, Indonesia. Cybium, 48(3), 247–250.

https://doi.org/10.26028/CYBIUM/2024-015

Hilgers, L., Herder, F., Hadiaty, R. K., & Pfaender, J.

(2018). Alien attack: Trophic interactions of flower-

horn cichlids with endemics of ancient Lake Matano

(Sulawesi, Indonesia). Evolutionary Ecology Research,

19(5), 575–590. http://www.evolutionary-ecology.

com/issues/v19/n05/jjar3112.pdf

Hoese, D., Mailautoka, K., & Ebner, B. (2019). Kuhlia mar-

ginata, silver flagtail. The IUCN Red List of Threatened

Species. https://doi.org/10.2305/IUCN.UK.2019-3.

RLTS.T11060A123377956.en

Hubert, N., Kadarusman, Wibowo, A., Busson, F., Caruso,

D., Sulandari, S., Nafiqoh, N., Pouyaud, L., Rüber,

L., Avarre, J.-C., Herder, F., Hanner, R., Keith, P., &

Hadiaty, R. K. (2015). DNA barcoding Indonesian

freshwater fishes: Challenges and prospects. DNA

Barcodes, 3(1), 144–169. https://doi.org/10.1515/

dna-2015-0018

IPBES. (2023). Summary for policymakers of the thema-

tic assessment report on invasive alien species and

their control of the Intergovernmental Science-Policy

Platform on Biodiversity and Ecosystem Services (H.

E. Roy et al., Eds.). IPBES Secretariat. https://doi.

org/10.5281/zenodo.7430692

Iqram, M., Litaay, M., Hasyim, Z., Moka, W. J. C., & Tinun-

gki, G. M. (2024). Combining eDNA metabarcoding

and conventional method in rapid screening of fish

diversity in Maros-Pangkep Global Geopark, South

Sulawesi, Indonesia. Biodiversitas, 25(2), 819–828.

https://doi.org/10.13057/biodiv/d250242

Ishihara, T., & Tachihara, K. (2008). Reproduction and

early development of a freshwater pipefish Microphis

leiaspis in Okinawa-jima Island, Japan. Ichthyologi-

cal Research, 55(4), 349–355. https://doi.org/10.1007/

s10228-008-0049-y

International Union for Conservation of Nature. (2025).

The IUCN Red List of Threatened Species. https://

www.iucnredlist.org

Iwasaki, W., Fukunaga, T., Isagozawa, R., Yamada, K.,

Maeda, Y., Satoh, T. P., Sado, T., Mabuchi, K., Takeshi-

ma, H., Miya, M., & Nishida, M. (2013). Mitofish and

mitoannotator: A mitochondrial genome database

of fish with an accurate and automatic annotation

pipeline. Molecular Biology and Evolution, 30(11),

2531–2540. https://doi.org/10.1093/molbev/mst141

Jacoby, D. M. P., Casselman, J. M., Crook, V., Delucia, M.,

Ahn, H., Kaifu, K., Kurwie, T., Sasal, P., Silfvergrip,

A. M. C., Smith, K. G., Uchida, K., Walker, A. M., &

Gollock, M. J. (2015). Synergistic patterns of threat

and the challenges facing global anguillid eel conser-

vation. Global Ecology and Conservation, 4, 321–333.

https://doi.org/10.1016/j.gecco.2015.07.009

Jenkins, A., Kullander, F. F., Tan, H. H., Sparks, J. S.,

& Pollom, R. (2017). Microphis leiaspis, barhead

pipefish. The IUCN Red List of Threatened Spe-

cies. https://doi.org/10.2305/IUCN.UK.2017-2.RLTS.

T169358A58318427.en

Keith, P., Allen, G., Lord, C., & Hadiaty, R. (2011).

Five new species of Sicyopterus (Gobioidei:

Sicydiinae) from Papua New Guinea and Papua.

Cybium, 35(4), 299–318. https://doi.org/10.26028/

CYBIUM/2011-354-004

18 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Keith, P., Hadiaty, R., Hubert, N., Busson, F., & Lord, C.

(2014). Three new species of Lentipes from Indone-

sia (Gobiidae). Cybium, 38(2), 133–146. https://doi.

org/10.26028/cybium/2014-382-004

Keith, P., & Lord, C. (2012). Tropical freshwater gobies:

Amphidromy as a life cycle. In R. A. Patzner, J. L.

Van Tassell, M. Kovačić, & B. G. Kapoor (Eds.), The

biology of gobies (pp. 243–277). CRC Press. https://

doi.org/10.1201/b11397-18

Keith, P., Lord, C., Busson, F., Sauri, S., Hubert, N., &

Hadiaty, R. (2015). A new species of Sicyopterus

(Gobiidae) from Indonesia. Cybium, 39(4), 243–248.

https://doi.org/10.26028/cybium/2015-393-005

Keith, P., Lord, C., & Maeda, K. (2014). Indo-Pacific sicydii-

ne gobies: Biodiversity, life traits and conservation.

Société Française d’Ichtyologie.

Keith, P., Mennesson, M. I., Sauri, S., Busson, F., Delrieu-

Trottin, E., Limmon, G., Nurjirana, Dahruddin, H.,

& Hubert, N. (2020). Giuris (Teleostei: Eleotridae)

from Indonesia, with description of a new species.

Cybium, 44(4), 317–329. https://doi.org/10.26028/

cybium/2020-444-003

Kennard, M., Brooks, S., Kerezsy, A., & Ebner, B. (2019).

Leiopotherapon unicolor. The IUCN Red List of

Threatened Species. e.T122906520A123382321.

https://doi.org/10.2305/IUCN.UK.2019-3.RLTS.

T122906520A123382321.en

Kottelat, M., Whitten, A. J., Kartikasari, S., & Wirjoatmod-

jo, S. (1993). Freshwater fishes of western Indonesia

and Sulawesi. Periplus.

Kottelat, M., & Whitten, A. J. (1996). Freshwater fishes of

western Indonesia and Sulawesi: Additions and correc-

tions. Periplus Editions.

Lagarde, R., Courret, D., Grondin, H., Faivre, L., & Ponton,

D. (2021). Climbing for dummies: Recommendations

for multi-specific fishways for the conservation of

tropical eels and gobies. Animal Conservation, 24(6),

970–981. https://doi.org/10.1111/acv.12699

Lohman, D. J., de Bruyn, M., Page, T., von Rintelen, K.,

Hall, R., Ng, P. K. L., Shih, H.-T., Carvalho, G. R., &

von Rintelen, T. (2011). Biogeography of the Indo-

Australian Archipelago. Annual Review of Ecology,

Evolution, and Systematics, 42, 205–226. https://doi.

org/10.1146/annurev-ecolsys-102710-145001

Lord, C., Brun, C., Hautecœur, M., & Keith, P. (2010).

Insights on endemism: Comparison of the duration of

the marine larval phase estimated by otolith micros-

tructural analysis of three amphidromous Sicyopterus

species (Gobioidei: Sicydiinae) from Vanuatu and New

Caledonia. Ecology of Freshwater Fish, 19(1), 26–38.

https://doi.org/10.1111/j.1600-0633.2009.00386.x

Lumbantobing, D. (2019a). Oryzias celebensis. The IUCN

Red List of Threatened Species. https://doi.org/10.2305/

IUCN.UK.2019-2.RLTS.T15579A90980558.en

Lumbantobing, D. (2019b). Telmatherina celeben-

sis. The IUCN Red List of Threatened Species.

https://doi.org/10.2305/IUCN.UK.2019-2.RLTS.

T21573A90980085.en

Lumbantobing, D., & Larson, H. (2019). Glossogobius fla-

vipinnis. The IUCN Red List of Threatened Species.

e.T40707A90982306. https://doi.org/10.2305/IUCN.

UK.2019-2.RLTS.T40707A90982306.en

Lynch, A. J., Elliott, V., Phang, S. C., Claussen, J. E., Harri-

son, I., Murchie, K. J., Steel, E. A., & Stokes, G. L.

(2020). Inland fish and fisheries integral to achie-

ving the Sustainable Development Goals. Nature

Sustainability, 3(8), 579–587. https://doi.org/10.1038/

s41893-020-0517-6

Maasri, A., Jähnig, S. C., Adamescu, M. C., Adrian, R., Bai-

gun, C., Baird, D. J., Batista-Morales, A., Bonada, N.,

Brown, L. E., Cai, Q., Campos-Silva, J. V., Clausnitzer,

V., Contreras-MacBeath, T., Cooke, S. J., Datry, T.,

Delacámara, G., De Meester, L., Dijkstra, K. B., Do,

V. T., … Worischka, S. (2022). A global agenda for

advancing freshwater biodiversity research. Ecolo-

gy Letters, 25(2), 255–263. https://doi.org/10.1111/

ele.13931

Madduppa, H., & Sani, L. M. I. (2020). Environmental DNA

sampling protocol. IPB University & Oceanogen.

Maeda, K., Kobayashi, H., Palla, H. P., Shinzato, C., Koya-

nagi, R., Montenegro, J., Nagano, A. J., Saeki, T.,

Kunishima, T., Mukai, T., Tachihara, K., Laudet, V.,

Satoh, N., & Yamahira, K. (2021). Do colour morphs

of an amphidromous goby represent different species?

Taxonomy of Lentipes (Gobiiformes) from Japan and

Palawan, Philippines, with phylogenomic approa-

ches. Systematics and Biodiversity, 19(8), 1080–1112.

https://doi.org/10.1080/14772000.2021.1971792

Maruyama, A., Nakamura, K., Yamanaka, H., Kondoh,

M., & Minamoto, T. (2014). The release rate of envi-

ronmental DNA from juvenile and adult fish. PLoS

ONE, 9(12), e114639. https://doi.org/10.1371/journal.

pone.0114639

McElroy, M. E., Dressler, T. L., Titcomb, G. C., Wilson, E.

A., Deiner, K., Dudley, T. L., Eliason, E. J., Evans, N.

T., Gaines, S. D., Lafferty, K. D., Lamberti, G. A., Li,

Y., Lodge, D. M., Love, M. S., Mahon, A. R., Pfrender,

M. E., Renshaw, M. A., Selkoe, K. A., & Jerde, C. L.

(2020). Calibrating environmental DNA metabar-

coding to conventional surveys for measuring fish

species richness. Frontiers in Ecology and Evolution, 8,

1–12. https://doi.org/10.3389/fevo.2020.00276

Mennesson, M. I., Bonillo, C., Feunteun, E., & Keith, P.

(2018). Phylogeography of Eleotris fusca (Teleostei:

Gobioidei: Eleotridae) in the Indo-Pacific area reveals

a cryptic species in the Indian Ocean. Conservation

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Genetics, 19(5), 1025–1038. https://doi.org/10.1007/

s10592-018-1063-x

Miesen, F. W., Droppelmann, F., Hüllen, S., Hadiaty, R. K.,

& Herder, F. (2016). An annotated checklist of the

inland fishes of Sulawesi. Bonn Zoological Bulletin,

64(2), 77–106.

Miya, M., Sato, Y., Fukunaga, T., Sado, T., Poulsen, J. Y.,

Sato, K., Minamoto, T., Yamamoto, S., Yamanaka, H.,

Araki, H., Kondoh, M., & Iwasaki, W. (2015). MiFish,

a set of universal PCR primers for metabarcoding

environmental DNA from fishes: Detection of more

than 230 subtropical marine species. Royal Society

Open Science, 2(7), 150088. https://doi.org/10.1098/

rsos.150088

Miya, M., Gotoh, R. O., & Sado, T. (2020). MiFish meta-

barcoding: A high-throughput approach for simul-

taneous detection of multiple fish species from

environmental DNA and other samples. Fisheries

Science, 86(6), 939–950. https://doi.org/10.1007/

s12562-020-01461-x

Moore, A. M., Jompa, J., Tassaka, A. C. M. A. R., Yasir, I.,

Ndobe, S., Umar, W., Gold, Z., & Barber, P. H. (2021).

Sharks and rays (Chondrichthyes) around Banggai

Island, Banggai MPA, Indonesia: Biodiversity data

from an environmental DNA pilot study. AACL Biof-

lux, 14(2), 725–745.

Moore, A. M., Yasir, I., Tassaka, A. C. M., Ndobe, S.,

Umar, W., Kadir, N. N., Putri, A. P., Yusuf, S., Bar-

ber, P. H., & Jompa, J. (2025). Perspectives from an

eDNA metabarcoding pilot project in the Banggai

MPA, Central Sulawesi, Indonesia. Bulletin of Mari-

ne Science, 101(1), 349–365. https://doi.org/10.5343/

bms.2023.0125

Morley, J., Buchanan, G., Mitchard, E. T. A., & Keane, A.

(2021). Implications of the World Bank’s environ-

mental and social framework for biodiversity. Con-

servation Letters, 14(1), 1–6. https://doi.org/10.1111/

conl.12759

Ndobe, S., Gani, A., Bakri, A. A., Adryani, D. T., Wuniarto,

E., Khartiono, L. D., Herjayanto, M., Hasan, V., &

Moore, A. M. (2022). First and recurrent records of

Stiphodon surrufus Watson & Kottelat, 1995 (Gobii-

formes, Gobiidae, Sicydiinae), a naturally rare amphi-

dromous goby, in Sulawesi, Indonesia. Check List,

18(2), 253–260. https://doi.org/10.15560/18.2.253

Ndobe, S., Nurdin, M. S., Hasanah, N., Putra, A. E.,

Mansyur, K., Nasir, M., Rabuna, M. L., & Moore,

A. M. (2023). DNA barcoding detects resurrected

taxon Giuris laglaizei (Sauvage, 1880) in Sulawesi,

Indonesia: Bolano Sau Lake payangka phylogeny,

phenotypic characters and implications for Giuris

spp. conservation. F1000Research, 11, 295. https://doi.

org/10.12688/f1000research.108970.2

Ndobe, S., Gani, A., Herjayanto, M., Bandjolu, K. P.,

Mokodongan, D. F., Khartiono, L. D., & Moore, A. M.

(2024). DNA barcode, sex ratio, and phenotypic cha-

racters of the Poso Lake (Sulawesi) endemic Mugilo-

gobius sarasinorum (Boulenger, 1897). Cybium, 48(3),

291–294. https://doi.org/10.26028/cybium/2024-022

Nedopil, C., Larsen, M., Chane-Yook, A., & Narain, D.

(2024). Catching up with climate priorities: Unders-

tanding multilateral development banks’ evolving

approach to biodiversity. Global Policy, 15(4), 660–

675. https://doi.org/10.1111/1758-5899.13403

Ng, H. H., & Low, B. W. (2019). Clarias batrachus. The

IUCN Red List of Threatened Species. https://

doi.org/10.2305/IUCN.UK.2019-2.RLTS.

T166613A1138872.en

Nurjirana, Burhanuddin, A. I., & Haris, A. (2019). Diversity

of penja fish (amphidromous goby) in Leppangan

River, West Sulawesi, Indonesia. AACL Bioflux, 12(1),

246–249. http://www.bioflux.com.ro/docs/2019.246-

249.pdf

Nurjirana, Keith, P., Burhanuddin, A. I., Haris, A., & Afri-

sal, M. (2021). DNA barcoding of two amphidromous

goby postlarvae (‘penja’) morphotypes from Mandar

River, West Sulawesi, Indonesia. Cybium, 45(3), 243–

249. https://doi.org/10.26028/cybium/2021-453-009

Nurjirana, N., Burhanuddin, A. I., Keith, P., Haris, A., &

Afrisal, M. (2022). Amphidromous goby postlar-

vae (penja) migration seasons and fisheries in West

Sulawesi, Indonesia. Biodiversitas Journal of Biological

Diversity, 23(1), 375–380. https://doi.org/10.13057/

biodiv/d230138

Nurjirana, Burhanuddin, A. I., Haris, A., Moore, A. M.,

Gani, A., Hasan, V., Motomura, H., Kobayashi, H.,

Haryono, H., & Mokodongan, D. F. (2024). Occurren-

ce and new data for the endemic amphidromous goby

Lentipes mekonggaensis in Central Sulawesi, Indone-

sia. Cybium, 48(4), 303–307. https://doi.org/10.26028/

cybium/2024-025

Oikonomou, A. (2021). Freshwater biogeography in a nuts-

hell. In E. Guilbert (Ed.), Biogeography: An integrative

approach of the evolution of living (pp. 219–243).

ISTE Ltd & John Wiley & Sons, Inc. https://doi.

org/10.1002/9781119882381.ch9

Ojaveer, H., Galil, B. S., Carlton, J. T., Alleway, H., Gou-

lletquer, P., Lehtiniemi, M., Marchini, A., Miller,

W., Occhipinti-Ambrogi, A., Peharda, M., Ruiz, G.

M., Williams, S. L., & Zaiko, A. (2018). Historical

baselines in marine bioinvasions: Implications for

policy and management. PLOS ONE, 13(8), e0202383.

https://doi.org/10.1371/journal.pone.0202383

Oliver, T. H., Heard, M. S., Isaac, N. J. B., Roy, D. B., Procter,

D., Eigenbrod, F., Freckleton, R., Hector, A., Orme, C.

D. L., Petchey, O. L., Proença, V., Raffaelli, D., Suttle,

K. B., Mace, G. M., Martín-López, B., Woodcock,

B. A., & Bullock, J. M. (2015). Biodiversity and res-

ilience of ecosystem functions. Trends in Ecology &

20 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Evolution, 30(11), 673–684. https://doi.org/10.1016/j.

tree.2015.08.009

Ottoni, F. P., South, J., Azevedo-Santos, V. M., Henschel, E.,

& de Bragança, P. H. N. (2023). Editorial: Freshwa-

ter biodiversity crisis: Multidisciplinary approaches

as tools for conservation. Frontiers in Environmen-

tal Science, 11, 1155608. https://doi.org/10.3389/

fenvs.2023.1155608

Pangemanan, N. P. L., Kepel, R. C., Bataragoa, N. E., Tum-

bol, R. A., Sahami, F. M., Lumenta, C., Salaki, M. S.,

Boneka, F. B., & Mantiri, D. M. H. (2020). Morpho-

metric and molecular analysis for identification of the

early stages of gobioid fishes in Poigar River estuary,

North Sulawesi, Indonesia. AACL Bioflux, 13(5),

2639–2646.

Paramata, A. R., Tresnati, J., Kadir, N. N., & Moore, A. M.

(2025). Native and introduced fish caught with gill-

nets in Limboto Lake, Gorontalo Province, Indonesia.

Fisheries and Aquatic Sciences, 28(4), 236–247. https://

doi.org/10.47853/FAS.2025.e21

Perry, W. B., Seymour, M., Orsini, L., Jâms, I. B., Milner,

N., Edwards, F., Harvey, R., de Bruyn, M., Bista, I.,

Walsh, K., Emmett, B., Blackman, R., Altermatt, F.,

Lawson Handley, L., Mächler, E., Deiner, K., Bik, H.

M., Carvalho, G., Colbourne, J., … Creer, S. (2024).

An integrated spatio-temporal view of riverine bio-

diversity using environmental DNA metabarcoding.

Nature Communications, 15(1), 4372. https://doi.

org/10.1038/s41467-024-48640-3

Pike, C., Crook, V., Jacoby, D., & Gollock, M. (2020). Angui-

lla interioris (Highlands long-finned eel). The IUCN

Red List of Threatened Species. e.T198972A176496665.

International Union for Conservation of Nature.

https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.

T198972A176496665.en

Porter, T. M., & Hajibabaei, M. (2018). Scaling up: A guide

to high-throughput genomic approaches for biodi-

versity analysis. Molecular Ecology, 27(2), 313–338.

https://doi.org/10.1111/mec.14478

Pyšek, P., Hulme, P. E., Simberloff, D., Bacher, S., Blackburn,

T. M., Carlton, J. T., Dawson, W., Essl, F., Foxcroft, L.

C., Genovesi, P., Jeschke, J. M., Kühn, I., Liebhold, A.

M., Mandrak, N. E., Meyerson, L. A., Pauchard, A.,

Pergl, J., Roy, H. E., Seebens, H., … Richardson, D.

M. (2020). Scientists’ warning on invasive alien spe-

cies. Biological Reviews, 95(6), 1511–1534. https://doi.

org/10.1111/brv.12627

Reid, A. J., Carlson, A. K., Creed, I. F., Eliason, E. J., Gell, P.

A., Johnson, P. T. J., Kidd, K. A., MacCormack, T. J.,

Olden, J. D., Ormerod, S. J., Smol, J. P., Taylor, W. W.,

Tockner, K., Vermaire, J. C., Dudgeon, D., & Cooke,

S. J. (2019). Emerging threats and persistent conser-

vation challenges for freshwater biodiversity. Biologi-

cal Reviews, 94(3), 849–873. https://doi.org/10.1111/

brv.12480

Righton, D., Piper, A., Aarestrup, K., Amilhat, E., Belpaire,

C., Casselman, J., Castonguay, M., Díaz, E., Dörner,

H., Faliex, E., Feunteun, E., Fukuda, N., Hanel, R.,

Hanzen, C., Jellyman, D., Kaifu, K., McCarthy, K.,

Miller, M. J., Pratt, T., … Gollock, M. (2021). Impor-

tant questions to progress science and sustainable

management of anguillid eels. Fish and Fisheries,

22(4), 762–788. https://doi.org/10.1111/faf.12549

Riley, I. J., DiBattista, J. D., Stewart, J., Schilling, H. T., &

Suthers, I. M. (2023). Using integrative taxonomy

to distinguish cryptic halfbeak species and interpret

distribution patterns, fisheries landings, and specia-

tion. Marine and Freshwater Research, 74(2), 125–143.

https://doi.org/10.1071/MF22048

Robin, F., Valen, S., Nomleni, A., Turnip, G., Luhulima,

M. Y., & Insani, L. (2023). Presence of non-native

freshwater fish in Indonesia: A review—risk and eco-

logical impacts. AACL Bioflux, 16(1), 66–79. http://

www.bioflux.com.ro/docs/2023.66-79.pdf

Robinson, C. V., Porter, T. M., Maitland, V. C., Wright, M.

T. G., & Hajibabaei, M. (2022). Multi-marker meta-

barcoding resolves subtle variations in freshwater

condition: Bioindicators, ecological traits, and tro-

phic interactions. Ecological Indicators, 145, 109603.

https://doi.org/10.1016/j.ecolind.2022.109603

Ruppert, K. M., Kline, R. J., & Rahman, M. S. (2019). Past,

present, and future perspectives of environmental

DNA (eDNA) metabarcoding: A systematic review

in methods, monitoring, and applications of global

eDNA. Global Ecology and Conservation, 17, e00547.

https://doi.org/10.1016/j.gecco.2019.e00547

Sahami, F. M., Kepel, R. C., Olii, A. H., Pratasik, S.

B., Lasabuda, R., Wantasen, A., & Habibie, S. A.

(2020). Morphometric and genetic variations of spe-

cies composition of nike fish assemblages in Goron-

talo Bay waters, Indonesia. Biodiversitas Journal of

Biological Diversity, 21(10), 4571–4581. https://doi.

org/10.13057/biodiv/d211015

Sato, Y., Miya, M., Fukunaga, T., Sado, T., & Iwasaki, W.

(2018). MitoFish and MiFish pipeline: A mitochon-

drial genome database of fish with an analysis pipeli-

ne for environmental DNA metabarcoding. Molecular

Biology and Evolution, 35(6), 1553–1555. https://doi.

org/10.1093/molbev/msy074

Serdiati, N., Arfiati, D., Widodo, M. S., Lelono, T. D.,

Ndobe, S., Mansyur, K., & Moore, A. M. (2020).

Perspectives on sustainable management of the Poso

Lake (Indonesia) endemic ricefish, Oryzias nigrimas

(Actinopterygii: Adrianichthyidae). Revista de Biolo-

gía Tropical, 69(1), 139–152. https://doi.org/10.15517/

rbt.v69i1.42404

Serdiati, N., Ndobe, S., Rosyida, E., & Moore, A. M.

(2023). Barcoding a glass eel migration: A snapshot

of taxa migrating upstream in the Palu River, Central

Sulawesi, Indonesia. IOP Conference Series: Earth and

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

Environmental Science, 1272(1), 012020. https://doi.

org/10.1088/1755-1315/1272/1/012020

Serdiati, N., Ndobe, S., Rosyida, E., Gani, A., Hermawan,

R., Nurdin, M. S., Sari, D. E., Herlina, S., Pawaro, M.

F. D., & Danty, A. R. (2024). Length-weight relation-

ship, condition factor and DNA barcoding of bonylip

barb, Osteochilus vittatus (Valenciennes, 1842) in

Dampelas Lake, Central Sulawesi, Indonesia. Omni-

Akuatika, 20(1), 23–37. https://doi.org/10.20884/1.

oa.2024.20.1.1111

Shao, X., Fang, Y., Jawitz, J. W., Yan, J., & Cui, B. (2019).

River network connectivity and fish diversity. Science

of the Total Environment, 689, 21–30. https://doi.

org/10.1016/j.scitotenv.2019.06.340

Shu, L., Ludwig, A., & Peng, Z. (2021). Environmen-

tal DNA metabarcoding primers for freshwater fish

detection and quantification: In silico and in tanks.

Ecology and Evolution, 11(12), 8281–8294. https://doi.

org/10.1002/ece3.7658

Sorensen, P. W. (Ed.). (2021). Biology and control of invasive

fishes. MDPI.

Stelbrink, B., Stöger, I., Hadiaty, R. K., Schliewen, U. K., &

Herder, F. (2014). Age estimates for an adaptive lake

fish radiation, its mitochondrial introgression, and an

unexpected sister group: Sailfin silversides of the Malili

Lakes system in Sulawesi. BMC Evolutionary Biology,

14(1), 94. https://doi.org/10.1186/1471-2148-14-94

Sugeha, H. Y. (2010). Recruitment mechanism of the tro-

pical glass eel genus Anguilla in the Poso Estuary,

Central Sulawesi Island, Indonesia. Jurnal Perikanan,

12(2), 86–100.

Takahashi, M., Saccò, M., Kestel, J. H., Nester, G., Camp-

bell, M. A., van der Heyde, M., Heydenrych, M. J.,

Juszkiewicz, D. J., Nevill, P., Dawkins, K. L., Bessey,

C., Fernandes, K., Miller, H., Power, M., Mousavi-

Derazmahalleh, M., Newton, J. P., White, N. E.,

Richards, Z. T., & Allentoft, M. E. (2023). Aquatic

environmental DNA: A review of the macro-orga-

nismal biomonitoring revolution. Science of the Total

Environment, 873, 162322. https://doi.org/10.1016/j.

scitotenv.2023.162322

Tauli, M., Garcia, M. P., Podeque, J. R., Signey, L., Sarmien-

to, K., & Santos, M. (2022). First record of the goby

Lentipes armatus Sakai and Nakamura, 1979 (Teleos-

tei: Gobiidae) in the Amburayan River in Benguet,

Luzon Island, Philippines. The Philippine Journal

of Fisheries, 29(1), 78–81. https://doi.org/10.31398/

tpjf/29.1.2020C0015

Tedesco, P. A., Beauchard, O., Bigorne, R., Blanchet, S.,

Buisson, L., Conti, L., Cornu, J.-F., Dias, M. S., Gre-

nouillet, G., Hugueny, B., Jézéquel, C., Leprieur, F.,

Brosse, S., & Oberdorff, T. (2017). A global database

on freshwater fish species occurrence in draina-

ge basins. Scientific Data, 4(1), 170141. https://doi.

org/10.1038/sdata.2017.141

Tembo, J. K., Kihia, C. M., Kitaka, N. K., Brien, G. O.,

Hanzen, C., Mbaru, E., & Wanjiru, K. C. (2023).

Diet and trophic interactions between catadromous

eels and sympatric fish in Kenyan east flowing river

systems. Western Indian Ocean Journal of Marine

Science, 22(2), 87–106. https://doi.org/10.4314/wio-

jms.v22i2.7

Thinh, D. V., Van, N. S., Nguyen, T. H. T., & Lumban-

tobing, D. (2020). Barbonymus gonionotus, silver

barb. The IUCN Red List of Threatened Species.

https://doi.org/10.2305/IUCN.UK.2020-3.RLTS.

T166914A89799952.en

Torres, A. G., Leander, N. J. S., Kesner-Reyes, K., Ballad,

E. L., Ame, E. C., Palla, H. P., Angeles, I. J. P., Gon-

zalez, J. C. T., Reyes, A. T., Gaerlan, R. S. P., Guino-o,

R. S. I., & Cecilio, M. A. F. (2021). Leiopotherapon

plumbeus. The IUCN Red List of Threatened Spe-

cies. https://doi.org/10.2305/IUCN.UK.2021-1.RLTS.

T162161619A162161646.en

Triyanto, T., Haryani, G. S., Lukman, Wibowo, H., Ali, F.,

Hidayat, Sulawesty, F., Setiawan, F. A., Triwisesa, E.,

Dwinovantyo, A., Riyanto, M., Samir, O., & Nafis-

yah, E. (2021). Perspective plan for sustainable eel

management in Lake Poso, Central Sulawesi. E3S Web

of Conferences, 322, 05014. https://doi.org/10.1051/

e3sconf/202132205014

Tweedley, J. R., Bird, D. J., Potter, I. C., Gill, H. S., Miller,

P. J., O’Donovan, G., & Tjakrawidjaja, A. H. (2013).

Species compositions and ecology of the riverine

ichthyofaunas in two Sulawesian islands in the bio-

diversity hotspot of Wallacea. Journal of Fish Biology,

82(6), 1916–1950. https://doi.org/10.1111/jfb.12121

Vari, R. P., & Hadiaty, R. K. (2012). The endemic Sulawesi

fish genus Lagusia (Teleostei: Terapontidae). Raffles

Bulletin of Zoology, 60(1), 157–162.

Wang, B., Jiao, L., Ni, L., Wang, M., & You, P. (2024). Brid-

ging the gap: The integration of eDNA techniques

and traditional sampling in fish diversity analysis.

Frontiers in Marine Science, 11, 1289589. https://doi.

org/10.3389/fmars.2024.1289589

Wantania, L. L., Koppetsch, T., Möhring, J., Miesen, F. W.,

Wowor, D., Boneka, F., & Herder, F. (2024). Sulawesi

stream fish communities depend on connectivity and

habitat diversity. Journal of Fish Biology, 105(4), 1–18.

https://doi.org/10.1111/jfb.15944

Williams-Subiza, E. A., & Epele, L. B. (2021). Drivers

of biodiversity loss in freshwater environments: A

22 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e20263754, enero-diciembre 2026 (Publicado Jul. 02, 2026)

bibliometric analysis of the recent literature. Aqua-

tic Conservation: Marine and Freshwater Ecosystems,

31(9), 2469–2480. https://doi.org/10.1002/aqc.3627

WoRMS Editorial Board. (2025). World Register of Marine

Species [Database]. https://doi.org/10.14284/170

Xu, Y., Huo, X., Chen, X., Wang, Z., Zhou, M., Zhu, J., Yan,

R., & Cai, Y. (2024). Comparing the performance

variability of different eDNA primers in fish moni-

toring. Watershed Ecology and the Environment, 6(5),

165–175. https://doi.org/10.1016/j.wsee.2024.08.003

Yanuarita, D., Inaku, D. F., Nurdin, N., Rahim, S. W.,

Kudsiah, H., Parawansa, B. S., Rukminasari, N.,

Irmawati, & Moka, W. (2020). Aquatic invasive spe-

cies distribution within Wallace region: A preli-

minary review. IOP Conference Series: Earth and

Environmental Science, 564(1), 012038. https://doi.

org/10.1088/1755-1315/564/1/012038

Zhu, T., Sato, Y., Sado, T., Miya, M., & Iwasaki, W. (2023).

MitoFish, MitoAnnotator, and MiFish pipeline:

Updates in 10 years. Molecular Biology and Evolution,

40(3), 1–5. https://doi.org/10.1093/molbev/msad035