Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

Molecular and morphological evidence reveals two fish species

of Giuris (Eleotridae: Gobiiformes) in Sulawesi, with

a range extension of G. aporocephalus

Arfiani Rizki Paramata1; https://orcid.org/0000-0002-9619-2494

Joeharnani Tresnati2*; https://orcid.org/0000-0002-4818-406X

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

Abigail Mary Moore3; https://orcid.org/0000-0002-4122-3740

1. Fisheries Resources Management Department, Faculty of Marine and Fisheries Technology, Gorontalo State University,

Gorontalo, Indonesia; arfiani@ung.ac.id

2. Fishery Resources Management Department, Faculty of Marine Science and Fisheries, Hasanuddin University,

Makassar, Indonesia; Jtresnati@unhas.ac.id (*Correspondence), nadiarti@unhas.ac.id

3. Graduate School, Hasanuddin University, Makassar, Indonesia; abigail@pasca.unhas.ac.id

Received 05-XII-2024. Corrected 18-V-2025. Accepted 18-III-2026.

ABSTRACT

Introduction: Gobies of the freshwater or amphidromous eleotrid genus Giuris (Sauvage 1880) are widespread

in tropical rivers and lakes. Eight species are recognized to date through extensive taxonomic revision of the spe-

cies complex G. margaritaceus and remain challenging to identify based solely on morphological characteristics.

Objective: The aim of this study was to clarify the Giuris species occurring in three lakes along the Northern arm

of Sulawesi Island in the Wallacea bioregion (Limboto, Tondano and Bolano Sau) using an integrated molecular

(DNA barcoding) and morphological approach.

Methods: Giuris specimens were collected from the three lakes, and their morphological characters were record-

ed. Before voucher specimen preservation, fin clippings were preserved in 96 % ethanol for genetic analysis. DNA

barcodes were produced through DNA extraction, PCR (primers Fish F1, Fish R1) and Sanger sequencing of

Cytochrome C Oxidase Subunit I Mitochondrial DNA (COI mtDNA) nucleotide sequences.

Results: The DNA barcodes (615 bp) nested in two clades: G. laglaizei (Bolano Sau Lake) and G. aporocephalus

(all three lakes). Morphometric and meristic data were consonant with DNA barcode species assignments.

Conclusions: Our study confirms that the fish known locally as payangka in Limboto and Tondano Lakes belong

to G. aporocephalus, while those from Bolano Sau Lake correspond to G. aporocephalus and G. laglaizei. This

study also expands the known range of G. aporocephalus, with five Giuris species now confirmed from Tomini

Bay watersheds.

Key words: Wallacea; DNA barcoding; morphology; amphidromy; Tomini Bay.

RESUMEN

Evidencia molecular y morfológica revela dos especies de peces Giuris (Eleotridae: Gobiiformes)

en Sulawesi, con extensión del rango de G. aporocephalus

Introducción: Los peces gobios del género eleótrido dulceacuícola o anfidromo Giuris (Sauvage 1880) están

ampliamente distribuidos en ríos y lagos tropicales. Las ocho especies identificadas hasta la fecha mediante

https://doi.org/10.15517/xxxxxxx

GENETICS

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INTRODUCTION

The gobies (Gobiiformes) are a highly

diverse taxonomic group of marine, freshwater

and diadromous fishes found throughout the

Indo-Pacific region and the Indo-Malay islands

(Keith & Lord, 2012; Larson et al., 2014). The

phylum Gobiiformes comprises 13 currently

recognized families, including the Gobiidae (>

167 genera and > 1 432 species) and Eleotridae

(> 36 genera, > 215 species) (Fricke et al., 2025;

Froese & Pauly, 2025). Typically, amphidro-

mous gobies contribute most to the diversity

of fish communities in the Indo-Pacific and

the Caribbean insular systems, and have the

highest levels of endemism (Keith, 2002; Keith,

2003; Lim et al., 2002; Marquet et al., 1999),

although many detailed aspects of the biologi-

cal cycle and the factors driving evolution in

amphidromous gobies remain poorly under-

stood (Keith & Lord, 2012).

Typically, amphidromous like most ele-

otrids, gobies of the genus Giuris Sauvage 1880

are found in rivers (mostly estuarine and lower

reaches) and lakes, often associated with aquatic

vegetation and rocky or gravel bottoms (Keith

et al., 2020). The adults of some of the larger

diadromous gobies are also locally important

as food fish, particularly the gudgeons (Eleotri-

dae) generally known as payangka (or payangga

in some regions) in Sulawesi, that can reach at

least 16 cm (Makmur et al., 2019; Putra et al.,

2020). These payangka populations are consid-

ered at risk, primarily from the introduction of

alien fish but also from environmental degra-

dation and, due to their organoleptic qualities,

from fishing pressure (Paramata et al., 2025;

Syafei, 2017).

The genus Giuris has a complicated taxo-

nomic history; all previously described taxa

assigned to the genus Giuris were synonymized

as G. margaritaceus Valenciennes 1837 around

50 years ago (Akihito & Meguro, 1974; Keith

et al., 2020). Giuris has been assigned the

masculine gender (Kottelat, 2013); however,

the grammatical gender of this genus has been

inconsistent, with both G. margaritaceus and

G. margaritacea appearing in the literature,

along with at least 12 other synonyms (Froese

& Pauly, 2025). Despite external morphological

similarities, Kottelat (2013) argued that Giuris

likely comprised multiple cryptic species, based

on the extensive distribution and variability of

this taxon. Recent taxonomic research integrat-

ing classical morphological and molecular data

have confirmed the species complex hypothesis

una extensa revisión taxonómica del complejo de especies G. margaritaceus siguen siendo difíciles de identificar

morfológicamente.

Objetivo: El propósito de este estudio fue identificar las especies de Giuris presentes en tres lagos situados a lo

largo del brazo norte de la isla de Sulawesi en la bioregión de Wallacea (Limboto, Tondano y Bolano Sau), utili-

zando un enfoque integrado de códigos de barras de ADN y caracteres morfomerísticos.

Métodos: Se recolectaron especímenes de Giuris en los tres lagos y se registraron los caracteres morfomerísticos.

Antes de preservar los especímenes como ejemplares de referencia, se tomaron muestras de aletas, las cuales fue-

ron preservadas en etanol al 96 % para análisis genéticos. Los códigos de barras de ADN se obtuvieron mediante

extracción de ADN, PCR (usando los cebadores Fish F1 y Fish R1) y secuenciación Sanger de las secuencias de

nucleótidos del ADN mitocondrial de la subunidad I del citocromo C oxidasa (COI mtDNA).

Resultados: Los códigos de barras de ADN (615 bp) formaron dos clados: G. laglaizei (lago Bolano Sau) y G. apo-

rocephalus (en los tres lagos). Los caracteres morfomerísticos fueron congruentes con las asignaciones de especies

basadas en los códigos de barras de ADN.

Conclusiones: Nuestro estudio confirma que los peces conocidos localmente como payangka en los lagos

Limboto y Tondano pertenecen a la misma especie, mientras que en el lago Bolano Sau el payangka incluye dos

especies de Giuris. Este estudio también amplía el rango conocido de G. aporocephalus, con cinco especies de

Giuris ahora confirmadas en las cuencas hidrográficas de la Bahía de Tomini.

Palabras clave: Wallacea; código de barras de ADN; Giuris; morfomerística; anfidromía; Bahía de Tomini.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

and described or re-described eight Giuris spe-

cies to date (Keith & Mennesson, 2020; Keith et

al., 2020; Ndobe et al., 2023).

The largest semi-enclosed bay in the world,

Tomini Bay (also known as the Gulf of Tomi-

ni) is bounded by the Southern coast of the

Northern arm of Sulawesi, the Northern coast

of Central Sulawesi, and to the West by the

“neck” joining them together (Fig. 1). Payangka

(Giuris spp.) are present in at least three lakes:

Bolano Sau in Central Sulawesi Province, Lim-

boto Lake in Gorontalo Province, and Tondano

Lake in North Sulawesi Province. Three species

Giuris were identified to the South of Tomini

Bay by a major Indonesia-wide study (Keith et

al., 2020), and a DNA barcoding study revealed

that the payangka in Bolano Sau lake, identi-

fied as G. laglaizei (Ndobe et al., 2023), is not

the same species as the payangka in Limboto

and Tondano.

There is anecdotal historical evidence

that the Giuris in Tondano Lake were intro-

duced over 100 years ago from Limboto Lake

(Soeroto, 1988), so it is likely that the same

species will be present in these two lakes. It is

also possible that more than one species may be

present in each lake. The aim of our study was

therefore to elucidate the Giuris species present

in the three main lakes on the Northern arm of

Sulawesi (Limboto, Tondano and Bolano Sau)

using an integrated morphological and molecu-

lar (DNA barcoding) approach. These data will

enrich DNA barcoding databases, inform man-

agement at the local level in three Indonesian

provinces, and provide input for conservation

assessments at local to global scales.

MATERIAL AND METHODS

Specimen collection and preservation:

Specimens of the genus Giuris were collected

on 13-18 August 2023 from three lakes on the

Northern arm of Sulawesi Island, Indonesia:

Limboto Lake, Gorontalo Province; Tondano

Lake, North Sulawesi Province; and Bolano Sau

Lake, Central Sulawesi Province (Fig. 1). The

Fig. 1. Research sites in the Northern arm of Sulawesi, Wallacea, Indonesia.

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

collection was authorized under research per-

mit No. 18057/UN4.15/PT.01.04/2024 issued

by Hasanuddin University and was approved

by the relevant provincial government agencies.

The specimens were obtained from local fish-

ermen using a throw net with mesh size 3.5”.

Each specimen collected was photographed

using an iPhone digital camera (Fig. 2) and

euthanized using clove oil following standard

protocols (Fernandes et al., 2017; Neiffer &

Stamper, 2009).

Each specimen was weighed (digital scales,

precision 0.1 g), labelled, and the total length

(TL) and standard length (SL) were measured

(digital calipers, precision 0.1 mm). A tissue

sample for genetic (DNA barcoding) analysis

was taken from the right ventral fin of each

specimen, cleaned with distilled water and

placed in a 2 ml cryotube containing 96 %

absolute ethanol. The tube was then labelled

with the same field sample code as the speci-

men: P1LL, P2LL, P3LL for samples from Lim-

boto Lake; P1TL, P2TL, P3TL for samples from

Tondano Lake; P1BL, P2BL for samples from

Bolano Sau Lake.

The specimens were then preserved follow-

ing the protocol developed for the Hasanuddin

University scientific collection (Omar et al.,

2021) with catalog numbers UNH24-NAS001-

UNH24-NAS008. After soaking in 4 % forma-

lin for 2-3 days, each specimen was rinsed in

clean water to remove formalin before con-

ducting the ethanol dehydration series (Suzuki

et al., 2012). The specimens were placed in 96

% ethanol to remove excess water for 3-7 days,

then transferred to glass jars filled with 70 %

alcohol which was changed periodically until

discoloration was minimal.

Morphometric and meristic data: Where

relevant, counts and measurements were taken

from the left side of each specimen. Morpho-

metric measurements (Fig. 3A, Fig. 3B, Table 1)

were made with digital calipers in mm (preci-

sion 0.1 mm). Meristic counts included scale

counts (Fig. 3C) and fin spine/ray counts. Char-

acter codes used followed references (Keith &

Mennesson, 2020; Keith et al., 2020). All mor-

phometric traits except standard length (SL)

were expressed as dimensionless ratios (% SL).

DNA barcoding: Genomic DNA was

extracted from each sample using Geneaid

Gsync kits following the manufacturer’s pro-

tocols. DNA presence and quality was visual-

ized through electrophoresis on 2 % agar gel

soaked in the fluorescent dye ethidium bromide

and viewed under ultraviolet (UV) radiation.

The target cytochrome oxidase I mitochondrial

DNA (COI mtDNA) gene fragment (DNA bar-

code region) was amplified through polymerase

chain reaction (PCR) using the forward primer

Fish F1 (5’TCA ACC AAC CAC AAA GAC

ATT GGC AC-3’) and reverse primer Fish R1

(5’-TAG ACT TCT GGG TGG CCA AAG AAT

CA3’) (Ward et al., 2005).

PCR amplification was conducted (Bio-

systemsTM VeritiTM 96-Well Thermal Cycler,

Fig. 2. Freshly caught Giuris sp. specimen P1LL collected on 13 August 2023 from Limboto Lake, Gorontalo Province,

Indonesia.

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Thermo Fisher Scientific) using the following

PCR profile: predenaturation at 94 °C for 2

minutes; 40 cycles of denaturation at 94 °C for

45 seconds, annealing at 45 °C for 45 seconds,

and extension at 72 °C for 1.5 minutes; and final

extension at 72 °C for 10 minutes. The PCR

product (was visualized through electrophore-

sis (110 V for 20 minutes) on 2 % agar gel with

TAE buffer and GelRed™ stain; a 4 μl aliquot

of product was placed in each well. Sanger

sequencing (ABI 3500 Genetic Analyzer, Ther-

mo Fisher Scientific) was then performed on all

PCR products with clear electrophoresis bands.

The chromatogram (.abi) files were

imported into MEGA 11 (Tamura et al., 2021)

for quality control and analysis. The forward/

reverse sequences from each specimen were

cleaned, aligned using ClustalW performed

in MEGA 11 (Tamura et al., 2021), merged

and trimmed to produce a single nucleotide

sequence (DNA barcode). These vouchered

DNA barcode sequences were then deposited in

the NCBI GenBank repository with accession

numbers PQ409574-PQ409581 (Table 2).

Phylogenetic analysis: Sequence align-

ment, trimming and phylogenetic analy-

ses were performed in MEGA 11 (Tamura et

al., 2021). Homologous sequences from the

genus Giuris obtained using the NCBI Basic

Local Alignment Search Tool BLAST-n on-

line function were downloaded in FASTA for-

mat and aligned (ClustalW) with the Giuris

DNA barcodes obtained from this study and

a homologous vouchered eleotrid sequence

from a different genus (Oxyeleotris marmorata,

GenBank accession PQ637337, catalog num-

ber UNH24-NAS009, collected from Limboto

Lake). Sequences with 90 % or greater overlap

with the sequences from our specimens (Table

2) were included in evolutionary analyses using

the Kimura-2- Parameter (K2P) model (Kimu-

ra, 1980) in MEGA 11 (default settings) with 1

000 bootstrap test replicates (Felsenstein, 1985).

The Neighbor-Joining (NJ) method was applied

to the full data set in Table 2 (83 sequences, 643

nucleotide positions) and the Maximum Likeli-

hood method was applied to a reduced and

trimmed data set (77 sequences, 604 nucleotide

Fig. 3. Giuris sp. A. and B. morphometric and C. meristic characters used in this study.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

positions). The resulting phylogenetic trees

were exported as Newick files and edited in

the Interactive tree of life (iTOL) v5 (Letunic &

Bork, 2021).

The Giuris clades formed were identified

with assistance from Philippe Keith of the

French National Museum of Natural History.

RESULTS

Morphometric and meristic data: the

specimens showed considerable variation in

appearance (Fig. 4). The variability in the mor-

phological traits (Table 3) and meristic counts

(Table 4) of these specimens indicates the

presence of more than one species. Based on a

comparison with the most recent descriptions

or redescriptions of the eight currently recog-

nized species within the genus Giuris (Keith &

Mennesson, 2020; Keith et al., 2020), the most

likely species candidates were G. aporocephalus

Macleay, 1884, originally described from Aus-

tralia, and G. laglaizei Sauvage, 1880, originally

described from the Philippines.

Table 1

Morphometric characters measured on Giuris specimens from lakes in the Northern arm of Sulawesi.

No. Code Description

1 TL Total length

2SL Standard length

3 HL Head length

4 JL jaw length

5PDL Predorsal length

6PAL Preanal length

7 CPM Caudal peduncle height (minimum distance)

8BDD1 Body depth at first dorsal fin

9BDa Body depth at anus

10 O Eye diameter

11 Pect-L Pectoral fin length

12 SDFL Second dorsal fin length

13 CFL Caudal fin length

14 AFL Anal fin length

15 IO Interorbital length

16 LS Lateral line scale series (counted from upper pectoral base, or anteriormost scale along lateral midline, to

central hypural base)

17 TRF Transverse forward series (counted from the first scale anterior to the second dorsal fin, diagonally, anterior

and ventral to the middle of the abdomen or most ventral scale)

18 TRB Transverse backward series (counted from the first scale on the front to the second dorsal fin, diagonally,

on the back and ventral side to the base of the anal fin or most ventral scale)

19 ZZ Zig-zag series (scales on the narrowest part of the caudal peduncle counted from the most dorsal scale to

the most ventral scale in an alternating manner)

20 PD Pre-dorsal series (counted from scale directly anterior to first dorsal fin insertion to the anteriormost

midline scale)

21 D1 Anterior dorsal fin spines (Latin numbers) and rays (Arabic numbers)

22 D2 Posterior dorsal fin spines (Latin numbers) and rays (Arabic numbers)

23 P Pectoral fin spines (Latin numbers) and rays (Arabic numbers)

24 V Ventral fin spines (Latin numbers) and rays (Arabic numbers)

25 A Anal fin spines (Latin numbers) and rays (Arabic numbers)

26 C Caudal fin rays (Arabic numbers)

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Table 2

Nucleotide sequences used in the phylogenetic analyses.

No GenBank Accession Taxon given in

reference

Taxon based on

phylogenetic analysis

Country/

RegionbReference

Number(s) Verbatim taxona

1 AF391368 O. aporos O. aporos G. aporocephalus Australia (Thacker, 2003)

2AY722159, AY722160 O. aporos O. aporos G. aporocephalus Australia (Thacker & Hardman, 2005)

3 AY722161 O. aporos O. aporos G. aporocephalus Sulawesi * (Thacker & Hardman, 2005)

4HQ654732-HQ654738, HQ654740 O. aporos O. aporos G. laglaizei Philippines (Aquino et al., 2011)

5HQ682711, HQ682712 G. margaritacea G. margaritacea G. laglaizei Philippines (Aquino et al., 2011)

6JN021218, JN021219 G. tolsoni G. tolsoni G. tolsoni Philippines (Abdulmalik-Labe & Quilang, 2024)

7KU692503, KU692508, KU692513 G. margaritaceus G. margaritacea G. viator Java * (Dahruddin et al., 2017)

8KU692504-KU692506, KU692514 G. margaritaceus G. margaritacea G. tolsoni Bali * (Dahruddin et al., 2017)

9 KU944837 G. margaritaceus N/A G. tolsoni Taiwan (Chang et al., 2017)

10 MG407388-MG407392 G. margaritaceus G. margaritaceus G. laglaizei Philippines (Abdulmalik-Labe & Quilang, 2019)

11 MK572389 O. porocephalum O. porocephala G. viator Bangladesh (Rahman et al., 2019)

12 MW497105, MW497106, MW497109,

MW497111, MW497112, MW497116,

MW497125, MW497133, MW497145,

MW497146

Giuris sp. G. tolsoni G. tolsoni Lombok * (Keith et al., 2020)

13 MW497123 Giuris sp. G. tolsoni G. tolsoni Ampana * (Keith et al., 2020)

14 MW497113, MW497137, MW497139,

MW497147

Giuris sp. G. margaritaceus G. margaritaceus Ambon * (Keith et al., 2020)

15 MW497110, MW497117, MW497122 Giuris sp. G. margaritaceus G. margaritaceus Ampana * (Keith et al., 2020)

16 MW497141, MW497143 Giuris sp. G. margaritaceus G. margaritaceus Luwuk * (Keith et al., 2020)

17 MW497121 Giuris sp. G. margaritaceus G. margaritaceus Seram * (Keith et al., 2020)

18 MW497144cGiuris sp. G. viator G. viator Ambon * (Keith et al., 2020)

19 MW497108, MW497120 Giuris sp. G. viator G. viator Lombok * (Keith et al., 2020)

20 MW497135, MW497150cGiuris sp. G. viator G. viator Luwuk * (Keith et al., 2020)

21 MW497107, MW497114, MW497118,

MW497132, MW497136c, MW497149,

MW497151, MW497152, MW497153c

Giuris sp. G. viator G. viator Seram * (Keith et al., 2020)

22 OM674613 G. laglaizei G. laglaizei G. laglaizei Bolano Sau Lake * (Ndobe et al., 2023)

23 ON604188 G. margaritaceus N/A 2020-0156 14545

G. yahayai

Madagascar Unpublished 2022

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No GenBank Accession Taxon given in

reference

Taxon based on

phylogenetic analysis

Country/

RegionbReference

Number(s) Verbatim taxona

24 OQ386785 G. tolsoni G. tolsoni G. tolsoni Philippines (Bemis et al., 2023)

25 OQ386907 G. tolsoni G. tolsoni G. tolsoni Philippines (Bemis et al., 2023)

26 OQ788245-OQ788247, OQ788250 G. margaritaceus G. margaritaceus Giuris aporocephalus Limboto Lake* (Lamadi et al., 2023)

27 LC864454 G. tolsoni N/A G. tolsoni Japan Unpublished 2025

Giuris sequences obtained from this study and outgroup sequence

No. Specimen Code GenBank Accession UNHAS catalog number Collection site

Number Taxon Lake Coordinatesd

1 P1LL PQ409574 G. aporocephalus UNH24-NAS001 Limboto 0°35’02” N, 122°58’48” E

2 P2LL PQ409575 G. aporocephalus UNH24-NAS002 Limboto 0°35’02” N, 122°58’48” E

3 P3LL PQ409576 G. aporocephalus UNH24-NAS003 Limboto 0°35’02” N, 122°58’48” E

4 P1BL PQ409577 G. laglaizei UNH24-NAS004 Bolano Sau 0°27’05” N, 120°53’36” E

5 P2BL PQ409578 G. aporocephalus UNH24-NAS005 Bolano Sau 0°27’05” N, 120°53’36” E

6 P1TL PQ409579 G. aporocephalus UNH24-NAS006 Tondano 1°13’38” N, 124°53’49” E

7 P2TL PQ409580 G. aporocephalus UNH24-NAS007 Tondano 1°13’38” N, 124°53’49” E

8 P3TL PQ409581 G. aporocephalus UNH24-NAS008 Tondano 1°13’38” N, 124°53’49” E

9Outgroup PQ637337 Oxyeleotris marmorata UNH24-NAS009 Limboto 0°35’02” N, 122°58’48” E

a The taxon name given in the GenBank accession metadata. / b The sub-national region is given for accessions from Indonesian specimens (marked with *). / c Included in Neighbor

Join analysis but not in Maximum Likelihood analysis (insufficient overlap with sequences from this study). / d Approximate coordinates, given to the nearest second.

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The specimens in this study displayed

three color morphs. The first group (Fig. 4B,

Fig. 4D, Fig. 4E, Fig. 4F) have a greyish back-

ground along the dorsal ridge, with poorly-

defined and variable pale reddish and brownish

patterns on the flanks, becoming paler on the

belly. Some scales in lines running from the

pectoral fin base to the caudal peduncle along

and mostly close to the lateral line have a bluish

or greenish tinge, with orange to reddish spots

Fig. 4. Giuris specimens collected from lakes in the Northern arm of Sulawesi, Indonesia: A. G. aporocephalus (P1TL, SL =

mm; TL = mm); B. G. aporocephalus (P2TL, SL = mm; TL = mm); C. G. aporocephalus (P3TL, SL = mm; TL = mm); D. G.

aporocephalus (P1LL, SL = mm; TL = mm); E. G. aporocephalus (P2LL, SL = mm; TL = mm); F. G. aporocephalus (P3LL, SL

= mm; TL = mm); G. G. laglaizei (P1BL, SL = mm; TL = mm); H. G. aporocephalus (P2BL, SL = mm; TL = mm); codes: TL

= Tondano Lake; LL= Limboto Lake; BL = Bolano Sau Lake.

Table 3

Comparison of ten morphometric traits of eight Giuris sp. specimens from lakes in the Northern arm of Sulawesi and

reported ranges for eight Giuris species.

Specimen code/

species

Morphometric trait (ratio to standard length SL, in %, all measurements in mm) a

BDa BDD1 CPM PDL PAL CFL HL JL O IO

P1LL 22 28 17 50 67 12 30 10 8 11

P2LL 22 25 14 43 58 12 32 9 6 12

P3LL 20 23 13 44 58 14 30 9 6 13

P1TL 24 26 13 46 66 12 33 10 7 11

P2TL 26 28 13 43 64 12 32 10 4 12

P3TL 22 24 13 44 67 13 33 10 6 12

P1BL 31 27 18 47 64 13 37 13 5 0

P2BL 26 28 18 47 69 12 34 8 6 0

G. aporocephalusb21-26 20-25 13-16 44-51 62-67 22 31-36 9-11 5-7 12-16

G. laglaizei b 23-27 22-27 14-17 43-47 64-70 18 30-35 8-11. 5-6. 10-12.

G. laglaizei d15-27 10-16 27-40 9-15 20-31 4-8 3-6

G. viator bc 21-24 20-24 14-15 42-47 59-66 7-12 31-35 10-12 6-8 10-14

G. margaritaceus bc 20 -24 20-25 13-16 43-39 59-66 18 30-35 10-11 6-8 12-14

G. tolsoni bc 16 -22 20-24 13-15 39-47 59-68 11 31-37 9-12 6-7 9-12

G. yahayai b26 - 36 26-39 16-20 44-50 60-68 11-17 31-36 9-12 4-6 14-16

G. charpini b20 -24 19-25 13-16 43-44 59-65 10-12 31-33 10-11 5-7 12-14

G. caussei b16 -21 22-25 14-16 50 63-64 6 37 10 4-6 13;-5

a BDa = Body depth at anus; BDD1 = Body depth at first dorsal fin; CPM = Caudal peduncle height; PDL = Predorsal length;

PAL = Preanal length; CFL= Caudal fin length; HL = Head length; JL= Jaw length; O = Eye diameter; IO = Interorbital length.

See Table 1 for definitions. The full data set is available on reasonable requests from the corresponding author. / b (Keith &

Mennesson, 2020) / c (Keith et al., 2020). / d (Ndobe et al., 2023).

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

on some scales on the flanks from the pectoral

fin base to the hypural base. These colors can

cause the fish to shine in some light conditions.

Top of head greyish, lateral part yellowish with

3 dark brown to black stripes radiating from the

eye to the cheeks and operculum, the highest

stripe continuing to the pectoral base which is

yellowish. Both dorsal fins and the anal fin are

greyish with a reddish stripe at the distal mar-

gin. Pectoral fins greyish to hyaline and pelvic

fins greyish to hyaline with a red distal margin.

Caudal fin brownish with a reddish base and

paler outer margin.

The second group (Fig. 4A, Fig. 4C, Fig.

4H) have a brownish to orange dorsal color,

orange to dark red or brown on the flanks

with bluish coloration along and parallel to the

lateral line from the pectoral fin base to the

caudal peduncle. The pinkish orang to reddish

tinged scales on the flanks have a dark red dot.

Belly bright yellow or orange at the isthmus,

head brownish on top, sides orange to bright

red with 3 dark red stripes radiating from the

eye to the cheeks and operculum, the highest

stripe continuing to the bright yellow pectoral

base, and lips orange. The specimen P2BL (Fig.

4G) had more similarities to the first group but

displayed several differences from both groups.

DNA Barcoding and phylogenetic analy-

sis: The DNA barcodes obtained from the eight

Giuris sp. specimens collected from three lakes

in the Northern arm of Sulawesi were 587-604

bp in length and submitted to GenBank as

accessions PQ409574-PQ409581. The BLAST

results identified AF391368 (Thacker, 2003)

as the closest accession for seven of our Giuris

sequences (PILL, P2LL, P3LL, PITL, P2TL,

P3TL, and PIBL), with around 99 % identity.

This sequence was deposited as Ophieleotris

aporos and is currently listed in GenBank as

Giuris margaritaceus. The closest accession for

Table 4

Meristic count of eight Giuris sp. specimens from lakes in the Northern arm of Sulawesi and ranges for eight Giuris species.

Meristic fin spine/ray and scale countsa

D1-D2 A P V C LS PD TRB TRF ZZ

P1LL VI-I.8 I.7 10 I.5 14 27 15 10 12 8

P2LL VI-I.10 I.9 13 I.5 15 29 16 11 13 9

P3LL VI-I.9 I.9 15 I.5 15 30 18 12 14 9

P1TL VI-I.8 I.7 12 I.5 15 27 15 8 11 8

P2TL VI-I.8 I.9 13 I.5 15 29 14 10 12 8

P3TL VI-I.8 I.9 14 I.5 15 30 18 11 13 9

P1BL VI-I.8 I.9 12 I.5 14 31 13 12 12 9

P2BL VI-I.8 1.9 15 I.5 15 32 13 12 11 9

G. aporocephalus bVI-I.8 I.9 14-15 I.5 13-14 28-31 15-18 8-10 11-13 8-10

G. laglaizei bVI-I.8 I.9 15 I.5 14-15 29-31 15-17 9-11 12-14 8-9

G. laglaizei dVI-I.8 I.8 13 I.5 15

G. viator cVI-I.8 I.9 14 I.5 13-14 28-32 14-16 10-12 14-15 9-10

G. margaritaceus cVI-I.8 I.9 14-15 I.5 13-15 28-31 15-17 9-10 13-14 9-10

G. tolsoni cVI-I.8 I.9 14 I.5 13-15 29-31 14-16 10-11 13-16 8-10

G. yahayai bVI-I.8-9 I.9 14 I.5 15 29-32 15-18 10-12 17-20 7-9

G. charpini bVI-I.8 I.8-9 13-14 I.5 13-14 27-29 13-15 9-11 13-14 8-9

G. caussei bVI-I.8-9 I.9 14-15 I.5 13 29 15-16 9 11-12 9

a Fin spines /rays: Roman numerals represent spines; Arabic numerals represent rays; D = dorsal fin; A = anal fin; P =

pectoral fin; V = ventral fin; C = caudal fin. Scale counts: LS = lateral line series; PD = pre-dorsal series; TRB = transverse

backward series; TRF = transvers forward series; ZZ = zig-zag series. See Table 1 for definitions. / b (Keith & Mennesson,

2020). / c (Keith et al., 2020). / d (Ndobe et al., 2023).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

one sequence (PIBL) with 99 % identity was

OM674613 (Ndobe et al. 2023), submitted

as G. laglaizei. The clustering patterns of the

phylogenetic analyses using the Neighbor Join

(not shown) and Maximum Likelihood (Fig. 5)

methods were consistent, with six major clades

and all sequences included in both analyses

resolving within the same clade.

The eight DNA barcode sequences from

our study were resolved within two of the six

clades in the phylogenetic tree incorporating

Giuris GenBank accessions. The DNA barcodes

Fig. 5. Maximum Likelihood phylogenetic tree of the genus Giuris based on 77 COI mtDNA barcode sequences (604 bp, x

1000 bootstrap test replicates) including 8 specimens from the Northern arm of Sulawesi, 68 Giuris GenBank accessions, and

the outgroup Oxyeleotris marmorata Bleeker, 1852. Highlight color: red = Limboto Lake; blue = Bolano Sau Lake; yellow =

Tondano Lake. Line color: red = G. laglaizei clade; blue = G. aporocephalus clade. The number of sequences in each clade is

given in parentheses.

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

from seven specimens (P1LL, P2LL, P3LL from

Limboto Lake; P1TL, P2TL, P3TL from Tonda-

no Lake; P2BL from Bolano Sau Lake) resolved

in the same clade as accessions of Ophieleotris

aporos from Australia (Thacker, 2003; Thacker

& Hardman, 2005). This clade (comprising

Giuris accessions PQ409574-PQ409580 from

this study) was identified as Giuris aporocepha-

lus (Philippe Keith, French National Museum

of Natural History, personal communication

2024). The Bolano Sau Lake specimen (code

P1BL, GenBank accession PQ409581) resolved

within the G. laglaizei clade.

The map in Fig. 6 shows the distribution of

records for the eight currently accepted Giuris

species. The closest georeferenced record for

G. aporocephalus is around 3 000 km from the

Northern arm of Sulawesi.

DISCUSSION

Molecular identification of Giuris spp.:

Until recently, all Giuris populations were clas-

sified as a single species, G. margaritaceus,

originally described as Eleotris margaritacea

Valenciennes 1837. This collective taxono-

my is still used in FishBase (Froese & Pauly,

2025) and the IUCN Red List (Larson, 2019),

although 17 invalid synonyms (including 4

misspellings) are listed in FishBase, and 14

are listed in Eschmeyer’s Catalog of Fishes

(Fricke et al., 2025). Of these, two (originally

ascribed to the genus Eleotris) have now been

resurrected as extant species-level taxa in the

genus Giuris: G. laglaizei and G. aporocepha-

lus. Current revisions recognize eight species

within the morphologically cryptic Giuris spe-

cies complex (Keith & Mennesson, 2020; Keith

et al., 2020). Our study generated eight COI

barcodes (PQ409574-PQ40981; one specimen

failed to amplify), complementing existing data

on the distribution of Giuris species. These bar-

codes reveal the presence of at least two Giuris

species in the Northern arm of Sulawesi: (1) G.

laglaizei (confirming the record by Ndobe et al.

(2023) from Lake Bolano Sau) and (2) G. aporo-

cephalus, a new record for Sulawesi, found in

all three lakes (Bolano Sau, Limboto, and Ton-

dano). This dual presence makes Bolano Sau a

new multi-species site (sympatric zone), while

conclusively resolving the taxonomic identity

of payangka in lakes Limboto and Tondano as

G. aporocephalus. Thereby, we resolve the taxo-

nomic question raised by Ndobe et al. (2023)

regarding these populations.

The genetic distances (measured in the

number of substitutions per site and represented

visually by the branch lengths in Fig. 5) between

Fig. 6. Georeferenced records for eight Giuris species, indicating a range extension of around 3 000 km Northwards for G.

aporocephalus.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

the six major (congeneric species) clades were

in the order of 0.01, while the inter-generic

distance with the outgroup was in excess of 0.1,

and the within-clade (intra-species) distances

were of the order of 0.001 or less. The excep-

tion was the group within the G. laglaizei clade

comprising sequences from Lanao Lake in the

Philippines (GenBank accessions MG407388-

MG407392) originally labelled as G. margari-

taceus by Abdulmalik-Labe & Quilang (2019).

The genetic distance between this sub-clade

and the G. laglaizei sequences from other sites

was around 0.01, indicating considerable genet-

ic diversity between populations within this

putative species. Nonetheless, in general the

inter-and intra-specific genetic distances in

the COI mtDNA barcode region are conso-

nant with other taxonomic groups, including

marine fishes (Wang et al., 2024), while within

the Gobiiformes, the interspecies distances are

within the range reported for the subfamily

Sicydiinae (Taillebois et al., 2014).

Notably, our study did not detect G. mar-

garitaceus sensu stricto (the taxon currently

recognized under this name after taxonomic

revisions). This supports the hypothesis pro-

posed by Ndobe et al. (2023) and Lamadi et al.

(2024) that previous reports of G. margaritaceus

from this region (including misapplied syn-

onyms G. margaritacea and O. aporos: Auliyah,

2019; Hasim et al., 2021; Lamadi et al., 2023;

Makmur et al., 2015; Makmur et al., 2019;

Nuha et al., 2020; Putra et al., 2020) likely repre-

sent misidentifications of other Giuris species,

stemming from historical taxonomic confla-

tions. However, absence of detection is not

proof of absence. Both G. margaritaceus and

G. tolsoni have been detected at other Sulawesi

sites, including Ampana on the South coast of

Tomini Bay (Keith et al., 2020). This distribu-

tion pattern suggests potential recruitment via

riverine connections between Tomini Bay and

our study lakes, implying that additional Giuris

species may occur in the region despite only

two being found here.

Integrative taxonomic identification and

range extension of G. aporocephalus: The

present study provides new records for Giuris

aporocephalus based on molecular and mor-

phological data. This species was previously

recorded at georeferenced sites in Australia,

Papua New Guinea and the Solomon Islands

(Keith & Mennesson, 2020). Our records of the

species represent an approximate range exten-

sion of around 1 100 km to the North (from

the Soloman Islands) and 2 000 km to the

West (from Queensland, Australia). This amp-

hidromous fish exhibits a patchy distribution

across Sulawesi, with potential occurrence in

areas between our study sites and its confirmed

range. However, these distributional patterns

require verification through molecular, mor-

phological and geographical evidence.

Overall, the morphological characteris-

tics of G. aporocephalus (morphometric ratios

based on standard length and meristic counts)

aligned with diagnostic ranges established in

the redescription by Keith & Mennesson (2020)

which validated its distinction from G. margari-

taceus. The key diagnostic characters (15 pecto-

rals rays, mid-body depth at anus 20-26 % SL,

and 11-13 scales in transverse forward series)

were consistently observed. Table 3 and Table

4 show several morphometric deviations from

Keith & Mennesson (2020) redescription. For

instance, in specimen P1LL, the body depth at

anus was (20 % SL vs 21-26 %); the body depth

at the first dorsal fin (28 % vs 20-25 %), caudal

peduncle height (17 % vs 13-16 %), head length

(30 % vs 31-36 %), eye diameter (8 % vs 5-7

%), and interorbital distance (11 % vs 12-16 %).

Similarly, in P2TL the specimen body depth at

the first dorsal fin exceeded the expected range

(28 % vs 20-25 %).

Meristic data (Table 4) largely align with

reported ranges for G. aporocephalus, though

some deviations occur (compared with ranges

by Keith & Mennesson, 2020): D2 rays (10 in

P2LL, 9 in P3LL vs. 8); A rays (7 in PILL/PITL

vs. 9); pectoral rays (10-13 vs. 14-15); C rays

(15 in five specimens vs. 13-14); lateral line

scales (27 in PILL/PITL vs. 28-31); PD scales

(14 in P2TL vs. 15-18); and TRB rows (12 in

PILL vs. 8-10). Longitudinal (11-13) and zigzag

(8-10) scale series matched expected ranges.

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

These minor differences likely represent natu-

ral intraspecific variation, contributing to our

understanding of morphological diversity in

G. aporocephalus. As genetically verified speci-

mens accumulate, character ranges may expand

beyond current reports (Keith & Mennesson,

2020; Keith et al., 2020). However, increasing

overlaps between species’ morphological ranges

underscore the necessity of integrative taxo-

nomic approaches for reliable identification.

The G. aporocephalus specimens studied

here exhibited two color morphs differing pri-

marily in flank coloration. Like other Giuris

gobies (Keith et al., 2020), they are considered

omnivorous, feeding mainly on filamentous

algae, small crustaceans and aquatic insects,

and inhabiting vegetated areas such as ponds,

lakes, swamps, and the lower reaches of rivers.

The observed variation in coloration and mor-

phometric/meristic data (including deviations

from published ranges) likely reflect phenotypic

plasticity in response to environmental factors

(Hossain et al., 2010; Mittelbach et al., 2014), a

common source of intraspecific diversity.

Sympatric Giuris species in Bolano Sau

Lake: DNA barcoding identified two Giuris

species in Northern Sulawesi lakes: G. aporo-

cephalus (all three lakes) and G. laglaizei (in

Lake Bolano Sau). The confirmation of G.

laglaizei (PDB1 specimen, Fig. 4G) is consis-

tent with a prior record (OM674613) from

this lake by Ndobe et al. (2023), while PDB2

specimen from Bolano Sau Lake represent G.

aporocephalus, demonstrating sympatry. Such

multi-species occurrences mirror patterns

observed elsewhere, including the Philippines

(Abdulmalik-Labe & Quilang, 2024), Australia

(Thacker, 2003; Thacker & Hardman, 2005),

and Indonesia (Dahruddin et al., 2017), includ-

ing Central Sulawesi (G. margaritaceus and

G. tolsoni in Luwuk/Ampana and G. viator in

Luwuk (Keith et al., 2020).

The P1BL specimen shows similar mor-

phological characters to specimens from Lake

Mainit (Philippines, Mindanao) identified as

G. laglaizei, as redescribed by Keith & Mennes-

son (2020). The G. laglaizei specimen found in

Bolano Sau Lake during this study (Fig. 4G)

has a dark brown dorsal region; brown flanks;

brownish-dark grey head; grayish belly; brown-

ish first dorsal fin; greyish pelvic fins; brown-

ish anal fin with 2-3 rows of grayish dots; and

brownish-dark grey caudal fin with two rows of

grayish dots.

The morphometric and meristic characters

of G. laglaizei (Table 3, Table 4) generally fall

within the ranges reported in Keith & Men-

nesson (2020), with slight differences found

in PDB1 specimen: body depth at anus (31 %

vs 21-26 %), head length (37 % vs 31-36 %),

body depth at the first dorsal fin (28 % vs 22-27

%), caudal peduncle depth (18 % vs 14-17

%), pectoral fin ray count (12 vs 13), caudal

fin ray count (14 vs 15), and predorsal scales

(13 vs 15-17).

The similarity between the two sympat-

ric Giuris species from Lake Bolano Sau (G.

aporocephalus and G. laglaizei) (Fig. 4G, Fig.

4H) explains both the lack of historical reports

recognizing multiple species and the single

local name “payangka”. A diagnostic difference

observed was the presence of reddish-yellow

flank spots in G. aporocephalus, absent in G.

laglaizei.

Implications for conservation and man-

agement: Taxonomy is often perceived as an

academic pursuit with limited practical appli-

cation, yet it can have extremely important

implications for conservation and resource

management, including fisheries (Agnarsson &

Kuntner, 2007). For the genus Giuris, the previ-

ous classification as a single widespread species

(G. margaritaceus) led to its “Least Concern”

IUCN assessment (Larson, 2019), as population

declines of this amphidromous species could

theoretically be offset by natural recruitment

across watersheds, or eventually through assist-

ed recruitment. However, the recognition of at

least eight distinct species, each with potentially

restricted ranges, demands revised conserva-

tion assessments, as some may now face extinc-

tion risks. Accurate management thus requires

precise species identification using integrative

taxonomy (DNA barcoding and morphology),

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026204, enero-diciembre 2026 (Publicado Abr. 08, 2026)

as advocated for cryptic taxa (Packer et al.,

2009; Yang et al., 2022).

Given the apparently patchy distribution,

widely separated populations (e.g., Sulawesi

and Philippine populations of G. laglaizei and

the Australian and Sulawesi populations of G.

aporocephalus) may represent separate stocks

or even evolutionarily significant units (ESUs)

sensu Moritz (1994), thus requiring separate

assessment at a sub-species level. Where con-

nectivity between populations is lacking or

extremely limited, several management units

may be needed within a single ESU (Hohen-

lohe et al., 2021) or at fine geographical scales

(Moore et al., 2021).

This research highlights the value of inte-

grative taxonomy approaches for exploring bio-

diversity. We confirmed that the fish known

locally as payangka in Limboto and Tondano

Lakes belong to the same species (G. aporo-

cephalus), while Bolano Sau hosts two Giuris

species (G. aporocephalus and G. laglaizei). Our

findings expand the known range of G. aporo-

cephalus and reveal five Giuris species now

confirmed in Tomini Bay watersheds, necessi-

tating updates to IUCN Red List and FishBase

classification to reflect this hidden diversity.

Ethical statement: The authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

This research was supported by the Cen-

tre for Education Financial Service (PUSLAP-

DIK Indonesia) and the Indonesia Endowment

Fund for Education (LPDP Indonesia) through

grant Number 202209091319.

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