Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

Insights from DNA barcoding endangered endemic Caridina shrimps

(Decapoda: Atyidae) in Lindu Lake, Indonesia

Novalina Serdiati1*; https://orcid.org/0000-0002-1905-555X

Muhammad Safir2; https://orcid.org/0000-0001-7972-6178

Akbar Marzuki Tahya2; https://orcid.org/0000-0001-8313-209X

Muh Saleh Nurdin1; https://orcid.org/0000-0003-0875-7211

Nur Hasanah1; https://orcid.org/0000-0002-3642-9517

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

1. Aquatic Resources Management Study Program, Faculty of Animal Husbandry and Fisheries, Tadulako University,

Palu 94118, Central Sulawesi, Indonesia; novalinaserdiati@untad.ac.id (*Correspondence), msalehnurdin@gmail.com,

nurhasanah.nura@gmail.com

2. Aquaculture Study Program, Faculty of Animal Husbandry and Fisheries, Tadulako University, Palu 94118, Central

Sulawesi, Indonesia; safirmuhammad@gmail.com, amtahya@gmail.com

3. Graduate School, Hasanuddin University, Makassar 90245, South Sulawesi, Indonesia; abigail@pasca.unhas.ac.id

Received 08-X-2024. Corrected 17-II-2025. Accepted 02-IX-2025.

ABSTRACT

Introduction: Humanity faces a multidimensional crisis with severe threats to often poorly known freshwater

biodiversity. Molecular tools, like DNA barcoding, can aid in biodiversity exploration, monitoring, and conser-

vation. Sulawesian Caridina shrimps are both ecologically significant and endangered yet remain understudied.

Objective: To contribute well-documented DNA barcode (COX1 mtDNA) sequences for three Caridina species

endemic to Lindu Lake.

Methods: We collected 224 Caridina shrimps from three sites around Lindu Lake in August (n = 73), and

October 2023 (n = 117), and July 2024 (n = 34). We measured and analyzed morphological traits as dimension-

less ratios of total length. We extracted DNA from six specimens (two per species) and COX1 mtDNA barcodes

obtained through PCR (primers jgLCO and jgHCO) and Sanger sequencing. We obtained homologous sequences

from GenBank (BLAST routine) and BOLD repositories for phylogenetic analyses. All molecular analyses were

performed in MEGA 11.

Results: One species was identified at each site. Morphological traits differ between species. Three haplotypes

with low divergence: one in Caridina kaili, one in Caridina linduensis, and one in both Caridina dali and C.

linduensis. Homologous sequences in GenBank and BOLD included very few Sulawesi endemic species; these

formed the closest sister clades to Lindu Lake Caridina.

Conclusions: We submitted to the GenBank repository the first reference DNA barcodes for each of the Lindu

Lake Caridina species. The poor resolution of COX1 mtDNA for Lindu Lake Caridina may be due to recent evo-

lutionary processes. Our study highlights the ongoing need for barcoding freshwater invertebrates, particularly

atyid shrimps from Sulawesi, in the Wallacea bioregion.

Keywords: atyid shrimps; Crustacea; cryptic species; lacustrine; mitochondrial DNA; Wallacea.

https://doi.org/10.15517/pqf2j611

GENETICS

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

INTRODUCTION

Humanity is facing a triple crisis with

synergistic challenges in biodiversity loss, cli-

mate change, and threats to human wellbeing

(Baldwin-Cantello et al., 2023). The current

rate of biodiversity loss has been described as

a 6th mass extinction (Ceballos et al., 2015).

Freshwater ecosystems cover less than 1 %

of the earth’s surface, but account for over

6 % of described species, and present com-

plex management challenges (Dudgeon et al.,

2006). Freshwater biodiversity is increasingly

at risk due to direct and indirect anthropogenic

threats, calling for urgent action from local to

global scales to “bend the curve of freshwater

biodiversity loss” (Albert et al., 2021; Reid et

al., 2019; Tickner et al., 2020). Data are cru-

cial in addressing this crisis and stemming or

reversing biodiversity losses, in particular to

support planning and action at appropriate

spatial and temporal scales; however, for many

aquatic taxa, appropriate data are often still

lacking or incomplete, especially for inverte-

brates (Ahmed et al., 2022; Dudgeon et al.,

2006; Reid et al., 2019; Strayer & Dudgeon,

2010; Tickner et al., 2020).

Sulawesi, formerly known as the Celebes, is

the largest island in Wallacea, formed through

the meeting of the Eurasian, Pacific and Austral-

asian geotectonic plates, and a hotspot for ter-

restrial and aquatic endemism (Michaux, 2010;

Stelbrink et al., 2012; Struebig et al., 2022). The

unique Sulawesian freshwater fauna includes

radiations of closely related species in both ver-

tebrate and invertebrate taxa (Mokodongan &

Yamahira, 2015; Schubart et al., 2008; Stelbrink

et al., 2014; Vaillant et al., 2013; von Rintelen,

Bouchet & Glaubrecht, 2007), including atyid

shrimps of the genus Caridina (von Rintelen,

von Rintelen, Meixner et al., 2007; von Rintelen

& Cai, 2009). The genus Caridina comprises

at least 763 species (De Grave et al., 2015)

with a complex biogeography (de Mazancourt

RESUMEN

Información obtenida del código de barras de ADN en camarones Caridina (Decapoda: Atyidae)

endémicos en peligro de extinción en el Lago Lindu, Indonesia

Introducción: La humanidad enfrenta una crisis multidimensional con graves amenazas a la biodiversidad de

agua dulce, a menudo poco conocida. Las herramientas moleculares, como el código de barras de ADN, pueden

ayudar en la exploración, monitoreo y conservación de la biodiversidad. Los camarones Caridina de Sulawesi son

ecológicamente significativos y están en peligro, pero siguen siendo poco estudiados.

Objetivo: Contribuir con secuencias bien documentadas de código de barras de ADN (COX1 mtDNA) para tres

especies de Caridina endémicas del Lago Lindu.

Métodos: Recolectamos 224 camarones Caridina de tres sitios alrededor del Lago Lindu en agosto (n = 73),

octubre 2023 (n = 117) y en julio 2024 (n = 34). Medimos y analizamos rasgos morfológicos como proporciones

adimensionales de la longitud total. Extrajimos ADN de seis especímenes (dos por especie) y obtuvimos códigos

de barras COX1 mtDNA mediante PCR (primers jgLCO y jgHCO) y secuenciación Sanger. Obtuvimos secuen-

cias homólogas de GenBank (rutina BLAST) y repositorios BOLD para análisis filogenéticos. Todos los análisis

moleculares se realizaron en MEGA 11.

Resultados: Identificamos una especie en cada sitio. Los rasgos morfológicos diferían entre las especies. Tres

haplotipos con baja divergencia: uno en C. kaili, uno en C. linduensis, y uno en C. dali y C. linduensis. Las secuen-

cias homólogas en GenBank y BOLD incluyeron muy pocas especies endémicas de Sulawesi; estas formaron los

clados hermanos más cercanos a Caridina del Lago Lindu.

Conclusiones: Enviamos al repositorio GenBank los primeros códigos de barras de ADN de referencia para cada

una de las especies de Caridina del Lago Lindu. La baja resolución del COX1 mtDNA para Caridina del Lago

Lindu puede deberse a procesos evolutivos recientes. Nuestro estudio destaca la necesidad de codificar genética-

mente los invertebrados de agua dulce, en particular los camarones átidos de Sulawesi, en la región biogeográfica

de Wallacea.

Palabras clave: camarones átidos; crustáceos; especies crípticas; lacustres; ADN mitocondrial; Wallacea.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

et al., 2019). By 2021 at least 39 Sulawesian

Caridina species were known (Dwiyanto et al.,

2021), with 26 species endemic to ancient lakes

(Poso and the Malili complex), including their

tributary rivers (Klotz et al., 2021; von Rintelen,

2011; von Rintelen & Cai, 2009). Although

data on their biology and ecology are limited,

these freshwater shrimps likely play important

ecological roles in their typically oligotrophic

habitats (von Rintelen & Cai, 2009).

Molecular (DNA-based) tools are increas-

ingly used for biodiversity exploration and

monitoring, supporting conservation planning

and action (Deiner et al., 2017; Gostel & Kress,

2022; Ruppert et al., 2019), including in fresh-

water ecosystems (Reid et al., 2019; Takahashi

et al., 2023). The use of molecular markers

(short nucleotide sequences) called DNA bar-

codes theoretically enable the identification of

organisms based on small samples taken from

the target organism (Gostel & Kress, 2022).

One widely used DNA barcode is a fragment of

the cytochrome oxidase I mitochondrial DNA

(COI or COX1 mtDNA) (Hebert & Gregory,

2005). Environmental DNA (eDNA) meth-

ods can detect the recent presence of organ-

isms from samples (e.g. water, soil, air) of the

environment where they live and have shed

their DNA (Ruppert et al., 2019). Molecular

methods can, inter alia, help detect cryptic

diversity within an accepted taxon (Keith et

al., 2020; Keith & Mennesson, 2020; Siriwut et

al., 2021). The results can help determine com-

munity composition (Hernawati et al., 2020),

change known species distributions (Moore

et al., 2019) and require revisions in their

conservation status (Ndobe et al., 2023). How-

ever, the accuracy and usefulness of barcoding

and metabarcoding in key biodiversity-related

tasks such as the identification and monitor-

ing of specific taxa or biotic communities, are

heavily dependent on the taxonomic coverage

and quality of data deposited in nucleotide

sequence databases (Hestetun et al., 2020) such

as the NCBI GenBank (Sayers et al., 2022) and

Barcode of Life Database (Bold) (Ratnasing-

ham & Hebert, 2007).

Research on Sulawesian Caridina shrimps

has contributed to the study of evolution, in

particular speciation and species radiation (de

Mazancourt et al., 2019; von Rintelen et al.,

2010; von Rintelen & Cai, 2009). Their attrac-

tive appearance and their behavior have also

made them popular in the global aquarium

trade (Calado et al., 2003; Herjayanto, Ndobe,

et al., 2019; Kiruba-Sankar et al., 2018). In

addition to fishing pressure for the ornamental

trade, many are threatened habitat degradation

or loss (De Grave et al., 2015; Klotz et al., 2021),

and at least one Caridina dennerli (von Rintelen

& Cai, 2009) may be extinct (von Rintelen,

2018). Despite their popularity and awareness

of their vulnerability, Sulawesian atyid shrimps

remain understudied. Specific gaps include data

on species distributions, incomplete exploration

of species complexes, and biodiversity explora-

tion, as evidenced by the ongoing discovery

and description of new species (Annawaty et

al., 2022; Klotz et al., 2021; Klotz et al., 2023).

Given the number of species and the complex-

ity of identification based on external morphol-

ogy alone, the use of molecular methods has the

potential to support the study of atyid shrimp

diversity and distribution patterns. However,

many Caridina species (including most Sulawe-

sian Caridina) lack reference sequences in the

NCBI and BOLD databases.

Lindu Lake has a surface area of around

34.5 km2, a maximum depth of around 72.6 m,

and lies within an enclave in the Lore Lindu

National Park in Central Sulawesi, Indonesia

(Annawaty & Wowor, 2015). The lake and

its watershed are within the UNESCO-recog-

nized Lore Lindu Biosphere Reserve (Coordi-

nation and Communication Management of

Lore Lindu Biosphere Reserve, 2020). Three

Caridina species have been described from the

lake (Annawaty & Wowor, 2015). Of these, C.

linduensis (Roux, 1904) has been assessed as

Critically Endangered (CR) under IUCN Red

List criteria (De Grave & Wowor, 2020), while

C. dali (Annawaty & Wowor, 2015) and C. kaili

(Annawaty & Wowor, 2015) have not been

assessed. The purpose of this study was to con-

tribute well-documented DNA barcode (COX1

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

mtDNA) sequences obtained from Caridina

spp. specimens collected from Lindu Lake.

MATERIAL AND METHODS

Material-shrimp specimens: The material

examined in this study comprised 224 atyid

shrimps of the genus Caridina collected around

Lindu Lake in Central Sulawesi, Indonesia

(01°16’-01°23’ S, 120°1-120°11’ E) on 21 August

(n = 73) and 14 October 2023 (n = 117), and on

6 July 2024 (n = 34). The specimens were cap-

tured using dip nets collected from three sites

(Fig. 1) and euthanized using clove oil at a con-

centration of 200 mg/L until fully unresponsive

(Coyle et al., 2007).

Material-DNA: In each year (2023 and

2024), body tissue from a specimen of each

of the three morphospecies collected was pre-

served in a 1.5 ml Eppendorf tube filled with 96

% absolute ethanol for genetic analysis. These

six samples were sent to the Bionesia Laborato-

ry in Denpasar, Indonesia for further analysis.

Morphological analysis: Each specimen

was measured (digital calipers, precision 0.1

mm), weighed (digital scales, precision 0.01

g) and identified based on morphological

characters with reference to species descrip-

tions (Annawaty & Wowor, 2015; Roux, 1904)

and other information on the Lindu shrimps

(Annawaty et al., 2016).

Fig. 1. Samples of Caridina shrimps from Lindu Lake and their collection sites. A. C. kaili (ID: UNTAD_2024_CS01). B. C.

dali (ID: UNTAD_2024_CS02). C. C. linduensis (ID: UNTAD_2024_CS03).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

Morphometric traits measured in mm

(digital calipers, precision 0.1 mm) were: total

length (TL), head length (HL), rostrum length

(RL), body length (BL) and telson length (TS).

Dimensionless ratios to total length (TL) were

calculated (%) and given as range and mean ±

standard deviation (SD). Statistical tests were

conducted in Microsoft Excel 365 at the 95

% confidence level. Mean values of each trait

were compared through analysis of variance

(ANOVA) followed by post-hoc Student’s t-test

if significant differences in mean values were

detected (p < 0.05).

DNA Barcoding and Phylogenetic Analy-

sis: Genomic DNA was extracted from 10

g sub-samples of muscle tissue taken from

each of the preserved (96 % absolute ethanol)

shrimp samples using DNeasy Tissue Kits (Qia-

gen) following the manufacturer’s protocol. The

extracted DNA samples were stored in a freezer

for further analysis (-20 °C).

A fragment of the cytochrome oxidase I

(COX1) mitochondrial DNA (mtDNA) gene

was amplified through polymerase chain reac-

tion (PCR) using the forward primer jgLCO

(5’-TIT CIA AYC AYA ARG AYA TTG G-3’)

and reverse primer jgHCO (5’-TAI ACY TCI

GGR TGI CCR AAR AAY CA-3’) (Geller et al.,

2013) on an Applied Biosystems™ 2720 Ther-

mal Cycler machine. Each PCR reaction tube

contained 25 µl comprising: 2 µl extracted DNA

template; 1.25 µl of each primer (10 mM), 9 µl of

ddH2O, and 12.5 µl Ready Mix. The PCR pro-

file comprised initial denaturation at 94 °C for

3 min; 38 cycles of denaturation at 94 °C for 30

s, annealing at 50°C for 30 s, extension at 72 °C

for 60 s; and final extension at 72 °C for 2 min.

PCR product presence was verified through

electrophoresis on 1 % agarose gel stained with

Nucleic Acid Gel Stain (GelRed®). Verified PCR

product was sent for Sanger sequencing at PT.

Genetika Science, Jakarta, Indonesia.

The Sanger sequencing trace files (.abi

files) were imported into MEGA 11 (Tamura

et al., 2021). The forward and reverse sequenc-

es produced from each sample were cleaned,

aligned, trimmed, and combined to produce

a consensus sequence (DNA barcode). The

six barcodes obtained were submitted to the

NCBI GenBank nucleotide repository (submis-

sion SUB14743114) and allocated GenBank

accession numbers PQ361195-PQ361200. The

genetic distance between the DNA barcodes

(Kimura, 1980), their protein and nucleotide

composition were calculated in MEGA 11.

The on-line NCBI BLAST routine (Altschul

et al., 1990) was used with the Organism option

set to Caridina (taxid: 96236). All 65 resulting

homologous sequences obtained were down-

loaded using the FASTA (aligned sequences)

option. COX1 DNA barcodes were downloaded

from the Barcode of Life Database (BOLD).

The BOLD Identification routine did not find

any records similar to the Lindu Lake Caridina

barcodes. The BOLD database had 118 pub-

licly available clusters or Barcode Index Num-

bers (BINS) containing 2 554 records from 20

countries. Of these, 2 452 had species names,

representing 197 species. BOLD BINS typically

represent low-level taxa (species or sub-species/

populations within a species). BINS with Indo-

nesia as a country of origin were downloaded

as FASTA files.

Phylogenetic analysis was conducted in

MEGA 11 (Tamura et al., 2021). Sequences

were aligned using the ClustalW (Thompson

et al., 1994) option. Phylogenetic trees repre-

senting inferred evolutionary history within

the genus Caridina were constructed using

the Neighbor-Join (NJ) algorithm (Saitou &

Nei, 1987) with 1 000 bootstrap test replicates

(Felsenstein, 1985). Evolutionary distances (as

number of base substitutions per site) were

computed using the Kimura 2-parameter meth-

od (Kimura, 1980). The first analysis included

65 GenBank accession nucleotide sequences

from 16 nominal species and covered a total of

673 positions. The second analysis included 141

selected BOLD accessions from 19 BINS (SMT

1) representing 12 nominal species and one

BIN labelled Caridina sp. and covered a total of

654 nucleotide positions. Representative trees

were exported as a Newick files and edited

using the interactive tree of life (iTOL) version

5 (Letunic & Bork, 2021).

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

RESULTS

Morphological traits: At each site, only

one species was identified (Fig. 1). The main

diagnostic character for distinguishing the

three Lindu Lake Caridina species is the rela-

tive length of the rostrum (Annawaty & Wowor,

2015). This is longest in C. linduensis, extending

well beyond the basal antennular peduncle seg-

ment; in C. dali, the rostrum barely overreaches

this segment, while in C. kaili the rostrum

barely reaches or falls short of the end of this

segment. A further diagnostic trait for C. kaili is

a stouter second pereiopod compared to C. dali,

while C. kaili also has the largest eggs, visible in

the ovigerous female specimen in Fig. 1A.

The typology of the collection sites cor-

responded with previous studies on the three

species (Annawaty et al., 2016; Annawaty &

Wowor, 2015), which describe C. kaili as an

obligate stream species and C. linduensis as a

truly lacustrine shrimp, while C. dali was found

in both stream and lacustrine habitat. They

also note that they never found two species at

the same site. In this study, all specimens from

the tributary river site (Fig. 1A) were identified

as C. kaili, consonant with its designation as an

obligate stream species. All specimens from the

first site along the shore of the lake were iden-

tified as C. dali (Fig. 1B), and those from the

second lakeshore site as C. linduensis (Fig. 1C).

Morphometric data on the specimens

caught show high variability between individu-

als within each species and considerable over-

lap between species (Table 1). Total length (TL)

of the 224 specimens collected ranged from 5.1

to 21.8 mm, with a weight range of 0.01-0.12 g.

The data in Table 1 indicates considerable

morphological plasticity within each of the

Lindu Lake Caridina species; C. kaili, consid-

ered an obligate stream-dwelling rather than

lacustrine shrimp, had the lowest intra-species

variation in each character. Each species dif-

fered significantly from the other two in at least

one character. The mean value of the diagnostic

character rostrum length (RL) relative to total

length (RL/TL) followed the order given in

the species descriptions (Annawaty & Wowor,

2015) for the length relative to the basal anten-

nular peduncle segment. However, there was

considerable overlap and intra-species variation

in this ratio (Fig. 2).

Table 1

Morphometric characters of Caridina spp. (n = 224) from Lindu Lake*.

N.° Species nTL (mm) HL/TL (%) RL/TL (%) BL/TL (%) TS/TL (%) W (g)

Range Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Range

1C. kaili 92 12.0-21.8 15.9 ± 1.9 26.02 ± 5.30a10.46 ± 2.22a60.22 ± 5.35a13.26 ± 3.12a0.01-0.12

2C. dali 49 5.1-20.7 11.66 ± 3.72 29.49 ± 8.06b10.78 ± 3.49a55.25 ± 10.90b14.22 ± 3.52ab 0.01-0.10

3C. linduensis 83 8.9-20.0 13.5 ± 2.5 29.91 ± 6.28b12.50 ± 4.22b61.55 ± 11.37a14.81 ± 2.90b0.01-0.10

* For each character (column), mean values with the same superscript letter do not differ significantly from each other (95 %

confidence level).

Fig. 2. Boxplot of the ratio rostrum length to total length

(RL/TL, in %) for Caridina spp. (n = 224) from Lindu Lake.

DNA barcodes: The DNA barcode

sequences obtained from Lindu Lake Caridina

shrimps belonging to three morphotypes were

673 bp long. These sequences were submitted

to the NCBI GenBank database (Submission

SUB14743114, accession numbers PQ361195-

PQ361200). The GC-AT ratio was 69 %

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

(59.138:40.862). The genetic distances between

sequences ranged from zero (0.0) to nearly

0.003 (Table 2).

There were three sites with single nucleo-

tide polymorphisms and 3 alleles within this

data set (Table 3). The nucleotide and protein

composition were similar for all six sequences

from the three putative species (Table 4).

The mutations were all silent, meaning that

the protein translation was the same for all

six barcodes.

LAST and phylogenetic reconstruction:

The BLAST-n routine output included very few

Table 2

Genetic distance (base substitutions per site) between COX1 mtDNA barcodes of Caridina shrimp from Lindu Lake.

N.° Sample ID GenBank

Accession

Sequence number

1 2 3 4 5

1 UNTAD_2023_CS01 PQ361195

2 UNTAD_2024_CS01 PQ361196 0.0000000

3 UNTAD_2023_CS02 PQ361197 0.0044726 0.0044726

4 UNTAD_2024_CS02 PQ361199 0.0029806 0.0029806 0.0014875

5 UNTAD_2023_CS03 PQ361198 0.0029806 0.0029806 0.0014875 0.0000000

6 UNTAD_2024_CS03 PQ361200 0.0029806 0.0029806 0.0014875 0.0000000 0.0000000

Table 3

Single nucleotide polymorphisms (SNPs) and alleles in 673 bp COX1 mtDNA barcodes of Caridina shrimp from Lindu Lake.

N.° Sample ID Sequence

Lab ID Morphospecies Nucleotide site number Allele

21 245 270

1 UNTAD_2023_CS01 BIOSUB280.001 Caridina kaili A G T 1

2 UNTAD_2023_CS02 BIOSUB280.002 C. dali G A A 2

3 UNTAD_2023_CS03 BIOSUB280.003 C. linduensis G A T 3

4 UNTAD_2024_CS01 BIOSUB237.004 C. kaili A G T 1

5 UNTAD_2024_CS02 BIOSUB237.003 C. dali G A T 3

6 UNTAD_2024_CS03 BIOSUB237.005 C. linduensis G A T 3

Table 4

Nucleotide and translated protein composition of Lindu Lake Caridina shrimp COX1 mtDNA barcodes.

No GenBank Accession Number Base composition (% of 673 positions)

T C A G

1PQ361195-PQ361197, PQ361199, PQ361200 34.324 21.545 24.814 19.316

3 PQ361198 34.175 21.545 24.963 19.316

Mean 34.299 21.545 24.839 19.316

Translated proteins (identical for all six sequences)

Protein %Protein %Protein %

Alanine 8.929 Isoleucine 6.250 Arginine 2.232

Cysteine 0.000 Lysine 0.000 Serine 7.589

Aspartic acid 3.125 Leucine 15.625 Threonine 5.357

Glutamic acid 0.893 Methionine 7.143 Valine 6.696

Phenylalanine 7.143 Asparagine 4.018 Tryptophan 2.679

Glycine 11.161 Proline 5.804 Tyrosine 1.786

Histidine 2.679 Glutamine 0.893 Total: 224 codons

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

homologous Caridina sequences. All sequences

with a high cover % (90-100 %) had a low iden-

tity (% ID range 77.81-86.03 %), well below

thresholds for species identity. Of these, the

closest match in the GenBank database (88.63

% ID) was deposited as Caridina boehmei

(accession OR765944) obtained from a domes-

ticated population of this Indonesian native

shrimp (Romadhona et al., 2024). Conversely,

all overlapping sequences with the highest high

ID scores (> 95 %) had low cover % (29 %). All

phylogenetic trees produced using this data set

had similar topography. The Lindu Lake bar-

code sequences were consistently nested in the

genus Caridina within a clade containing other

species from Sulawesi, while the next nearest

neighbors were mostly species known to occur

in Indonesia (Fig. 3).

The BOLD BINs with geolocation data

in Indonesia comprised many more Caridina

COX1 sequences compared to GenBank. Most

of these had a high cover % with the Lindu

Lake Caridina DNA barcode sequences. Most

of the species in the Indonesian BINs have geo-

located reports from Sulawesi in the GBIF data-

base (Global Biodiversity Information Facility,

2024); however, there were no BOLD Bins rep-

resenting named Sulawesi endemic species. The

neighbor-join phylogenetic tree shows three

major clades, one comprising the Lindu Lake

sequences (Fig. 4).

DISCUSSION

The sequences obtained are likely the first

reference barcode sequences for the Lindu

Fig. 3. Phylogenetic tree (Neighbor-Join algorithm, 1 000 bootstrap replicates) inferred from six Lindu Lake Caridina DNA

barcodes (COI mtDNA) and 65 homologous NCBI GenBank accessions (673 nucleotide positions). Branch color indicates

nominal species distribution: Red = Lindu Lake (this study); Blue = Sulawesi endemic; Green = Indonesian distribution;

Black = not reported from Indonesia.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

Lake Caridina species Caridina linduensis, C.

dali and C. kaili, and are the first to be made

publicly available. The identity with the closest

homologous atyid shrimp barcodes was well

below 90 %, whereas reported inter-specific

variation (p-distance in %) in the COI sequenc-

es ranged from 0.8 to 4.9 with 7.7 % diver-

gence over the entire Poso watershed clade for

Caridina shrimps in Poso Lake (von Rintelen,

von Rintelen, & Glaubrecht, 2007) and from

0.5 to 9.7 with 11.5 % divergence over the entire

Malili lake complex clade (von Rintelen, 2011).

Fig. 2 and Fig. 3 show multiple well-

separated clades for several nominal species, in

particular C. brachydactyla, C. cantonensis, C.

gracilipes, C. indistincta, C. papuana, C. sulawesi

and C. tenuirostris. This indicates a need for

further research integrating classic morphom-

eristic and molecular approaches to clarify the

taxonomy and distribution of species within the

genus Caridina. Conversely, in both trees the

COX1 sequences for the three putative species

present in Lindu Lake are grouped in a clade

with shorter internal (sub-clade) branches than

most single species clades. Furthermore, one

sub-clade contains identical sequences from

two morphospecies (C. dali and C. linduensis).

We conclude that the COX1 mtDNA frag-

ment used as a DNA barcode in this study could

not be used to reliably distinguish between the

three putative Caridina species present in Lindu

Lake. However, this marker can distinguish

Fig. 4. Phylogenetic tree (Neighbor-Join algorithm, 1 000 bootstrap replicates) inferred from six Lindu Lake Caridina

DNA barcodes (COX1 mtDNA) and 141 homologous COX1 sequences from 19 Indonesian BOLD BINS (654 nucleotide

positions). Different highlight colors represent the terminal sub-clades of the Lindu Lake clade (red branch).

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

Lindu Lake Caridina from all other congeners

for which homologous DNA barcodes are cur-

rently available in the BOLD and GenBank

sequence repositories, even with overlaps as

low as 29 % in terms of the gene region covered.

The usefulness of the COI/COX1 mtDNA

barcode (and mitochondrial DNA more gener-

ally) for successful species delineation is limited

in cases where recent speciation events have

occurred (Raupach & Radulovici, 2015). The

low interspecific genetic distances between the

three putative species in this study may be

due to recent evolutionary radiation. However,

incongruence between taxonomic units based

on mtDNA genotype and phenotype can result

from high phenotypic plasticity (Fritz et al.,

2007; González-Castellano et al., 2020), and

patterns of COX1 variability can vary consider-

ably between decapod taxa (Matzen-da Silva et

al., 2011). These results call for further research

with a range of molecular markers to deter-

mine whether the Lindu Lake morphotypes do

indeed correspond to species at a genetic level

and, if so, identify a suitable molecular marker

(alternative “barcode”) capable of reliably dis-

tinguishing them from one another. Ambiguity

in DNA barcodes for crustacean identification

are also reported for the genus Macrobrachium

(Rosyida et al., 2023). These cases highlight the

need to combine molecular and classical taxo-

nomic methods rather than relying fully on one

or the other, in particular to delineate taxa and

identify individuals.

With respect to other Sulawesi endemic

Caridina, homologous sequences were only

accessible for four out of around 40 species.

Therefore, no conclusions can be drawn regard-

ing the power of the COX1 mtDNA molecular

marker used in distinguishing the majority of

Sulawesian Caridina species from the Lindu

Lake Caridina or from each other. This under-

lines the ongoing need for DNA barcoding of

freshwater and diadromous invertebrates, as

well as fish (Hubert et al., 2015).

Threats to native Sulawesi freshwater

fish and invertebrates, especially the many

endemic species, include habitat degradation

or loss; unsustainable exploitation, most often

for human consumption or the ornamental

aquarium trade; and the introduction of alien

species (Annawaty et al., 2016; Herder et al.,

2012; Herder et al., 2022; Klotz & von Rintelen,

2013; Rahmawati et al., in press; Serdiati et al.,

2021; von Rintelen et al., 2012; von Rintelen,

von Rintelen, Meixner et al., 2007; von Rintelen

& Cai, 2009). Caridina shrimps are considered

particularly sensitive to pollution (von Rintelen

& Cai, 2009), vulnerable to predation by alien

invasive species (Herder et al., 2012; Herder et

al., 2022), and popular in the poorly document-

ed aquarium hobby (Calado et al., 2003; Udin,

2013; von Rintelen & Cai, 2009). While posing

a threat to wild populations, this popularity has

also raised public awareness of their plight (von

Rintelen et al., 2012). Although management

measures are clearly vital to conserve native/

endemic species in Sulawesi lakes and their

watersheds, no in situ conservation measures

are reported in studies on Caridina shrimps in

Lindu Lake and its tributaries (Annawaty et al.,

2016; De Grave & Wowor, 2020).

Lindu Lake lies within the Lore Lindu

UNESCO Biosphere Reserve (LLBR) estab-

lished in 1978; the lake and riparian villages

form an enclave in the Lore Lindu National

Park, gazette in 1999 (Nontji, 2016). Tourism

and fisheries have been promoted as livelihood

options for Lindu Lake villagers (Muhamad et

al., 2023), and alien fish have been introduced

for this purpose (De Grave & Wowor, 2020;

Herjayanto, Gani, et al., 2019). Conservation

management of the Lindu Lake watershed has

been complicated by complex socio-economic

and cultural dynamics, with considerable in-

migration and potential conflicts between eth-

nic groups, illegal logging, and other land-use

issues (Anugrahsari et al., 2020; Mappatoba et

al., 2017; Rahman et al., 2021). Initiatives to

conserve the biodiversity of Lindu Lake and its

watershed, in particular the Caridina shrimps

in this study, include a project funded in 2022

by the Mohamed bin Zayed Species Conserva-

tion Fund for the conservation of Caridina

linduensis (Mohamed Bin Zayed-Species Con-

servation Fund [MBZ-SCF], 2022). The overall

project goal was to develop a multi-pronged

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

conservation strategy, with proposed activities

including studies on the reproductive biol-

ogy and ecology of C. linduensis as well as

developing and deploying artificial spawning

grounds in the lake and educational/awareness-

building programs for local communities, and

the release of larvae. However, the source of

these larvae was not specified in (MBZ-SCF,

2022), and it is not clear how far the targets

were accomplished. The project report does

not mention the complexities raised by the

presence of more than one Caridina taxon, pos-

sibly as only C. linduensis (De Grave & Wowor,

2020) has been assessed under IUCN Red List

criteria and, as an endangered species, is there-

fore eligible for funding from the Mohamed

Bin Zayed-Species Conservation Fund . The

only information found on output or outcome

from this project is a blog page (Dwiyanto,

2022) which makes general recommendations

similar to those proposed for endemic species

conservation in other Sulawesi lakes such as

Poso Lake (Serdiati et al., 2021). In addition to

identifying and protecting or restoring critical

habitat, the threat from alien species could be

reduced through promoting the use of low-

value or unused (invasive) alien species for

human consumption or as fish/animal feed

(Rahmawati et al., in press). There is still a

need to develop awareness regarding endemic

species, seek and leverage local traditions that

could support conservation, and involve com-

munities and other stakeholders in developing

workable conservation strategies, while moni-

toring of conservation targets (e.g., Caridina

populations and habitat condition) is crucial to

evaluate effectiveness.

In addition to in-situ measures, ex-situ

conservation can support the conservation of

endangered aquatic species (Calado et al., 2003;

Stoeckle et al., 2022), including those found in

Sulawesi lakes (Herjayanto et al., 2023; Herjay-

anto, Gani, et al., 2019; Wicaksono et al., 2022).

Ex-situ husbandry and breeding of decapods

has been developed for supplying the ornamen-

tal trade (Calado et al., 2003), and could help

alleviate one of the greatest threats to Sulawesi

aquatic organisms, including Caridina shrimps.

Captive breeding could also enable re-stocking

of native/endemic species populations (Theiss-

inger et al., 2021). Such interventions should

follow national (Sadili et al., 2015) and inter-

national (e.g. IUCN) guidelines, in particular

with respect to biosecurity and genetic diver-

sity considerations (Bouwmeester et al., 2021;

Theissinger et al., 2021; Velle et al., 2025). In the

case of cryptic/morphologically similar species,

DNA barcoding can assist in ensuring prospec-

tive broodstock belong to the same species, and

molecular markers can also be used to evaluate

intra-species genetic diversity in captive popu-

lations and wild populations enhanced through

restocking (Fox et al., 2018; Roques et al., 2018;

Stoeckle et al., 2022), as well as supporting

traceability (Ng et al., 2016). With respect to the

Lindu Caridina species, ex-situ conservation

efforts include research on the husbandry as

a first step towards captive breeding of C. kaili

(Herjayanto, Ndobe, et al., 2019).

In conclusion, this study provides the first

reference DNA barcodes (COI/COX1 mtDNA)

for atyid shrimps from Lindu Lake in Central

Sulawesi, Indonesia with lengths of 673 bp.

The similarity between the sequences, obtained

from specimens identified as Caridina linduen-

sis, C. dali, and C. kaili based on external mor-

phomeristic traits, and the mismatch between

morphotypes and genotypes (alleles based on

three single nucleotide polymorphisms), may

indicate poor resolution of this commonly

used molecular marker for the genus Caridina,

and call for further research with a range of

molecular markers. More broadly, efforts are

needed to fill the gap in standard COI/COX1

DNA barcodes for Sulawesi endemic aquatic

invertebrates, in particular atyid shrimps of the

genus Caridina. Such research can contribute

to urgently needed conservation measures, in

particular the monitoring of wild populations

as well as ex-situ initiatives and traceability in

the international aquarium trade.

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

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e2025672, enero-diciembre 2025 (Publicado Set. 17, 2025)

followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowledg-

ments section. A signed document has been

filed in the journal archives.

See supplementary material

53v73n1-suppl. 1

ACKNOWLEDGMENTS

The authors acknowledge financial sup-

port from Tadulako University through the

2024 DIPA (Budget Implementation Form)

funds for Postgraduate Studies under Contract

Number 1298/UN28.16/AL.04/2024.

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