# Revisiting species boundaries and distribution ranges of Nemacheilus spp. (Cypriniformes: Nemacheilidae) and Rasbora spp. (Cypriniformes: Cyprinidae) in Java, Bali and Lombok through DNA barcodes: implications for conservation in a biodiversity hotspot

Nicolas Hubert, Daniel Lumbantobing, Arni Sholihah, Hadi Dahruddin, Erwan Delrieu-Trottin, Frederic Busson, Sopian Sauri, Renny Hadiaty,

Philippe Keith

► To cite this version:

Nicolas Hubert, Daniel Lumbantobing, Arni Sholihah, Hadi Dahruddin, Erwan Delrieu-Trottin, et al. Revisiting species boundaries and distribution ranges of Nemacheilus spp. (Cypriniformes: Nemacheilidae) and Rasbora spp. (Cypriniformes: Cyprinidae) in Java, Bali and Lombok through DNA barcodes: implications for conservation in a biodiversity hotspot. Conservation Genetics, 2019, 20 (3), pp.517-529. 10.1007/s10592-019-01152-w. hal-02549151

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1 Revisiting species boundaries and distribution ranges of *Nemacheilus* spp.

2 (*Cypriniformes: Nemacheilidae*) and *Rasbora* spp. (*Cypriniformes: Cyprinidae*) in

3 Java, Bali and Lombok through DNA barcodes: implications for conservation in a

4 biodiversity hotspot

5

6 Nicolas Hubert¹, Daniel Lumbantobing², Arni Sholihah¹,³, Hadi Dahruddin¹,², Erwan

7 Delrieu-Trottin¹,⁴, Frédéric Busson¹,⁵, Sopian Sauri², Renny Hadiaty², Philippe Keith⁵

8

9 ¹ UMR 226 ISEM (UM, CNRS, IRD, EPHE), Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier cedex 05, France.

10
11 ² Division of Zoology, Research Center for Biology, Indonesian Institute of Sciences
12 (LIPI), Jalan Raya Jakarta Bogor Km 46, Cibinong 16911, Indonesia.

13 ³ Instut Teknologi Bandung, School of Life Science and Technology, Bandung, Indonesia.

14 ⁴ Museum für Naturkunde, Leibniz-Institut für Evolutions und Biodiversitätsforschung an der Humboldt-Universität zu Berlin, Invalidenstrasse 43, Berlin 10115, Germany

16 ⁵ UMR 7208 BOREA (MNHN-CNRS-UPMC-IRD-UCBN), Muséum National d'Histoire Naturelle, 43 rue Cuvier, 75231 Paris cedex 05, France.

18

19

20 *Correspondence*: Nicolas Hubert, Institut de Recherche pour le Développement, UMR 226 ISEM (UM-CNRS-IRD-EPHE), Place Eugène Bataillon, CC 065, F-34095 Montpellier cedex 05, France.

23 E-mail: nicolas.hubert@ird.fr

24

25

26 Abstract

27 Biodiversity hotspots have provided useful geographic proxies for conservation efforts.

28 Delineated from a few groups of animals and plants, biodiversity hotspots do not reflect

29 the conservation status of freshwater fishes. With hundreds of new species described on

30 a yearly basis, fishes constitute the most poorly known group of vertebrates. This

31 situation urges for an acceleration of the fish species inventory through fast and reliable

32 molecular tools such as DNA barcoding. The present study focuses on the freshwater

33 fishes diversity in the Sundaland biodiversity hotspot in Southeast Asia. Recent studies
evidenced large taxonomic gaps as well as unexpectedly high levels of cryptic diversity, particularly so in the islands of Java and Bali. The Cypriniformes genera *Rasbora* and *Nemacheilus* account for most of the endemic species in Java and Bali, however their taxonomy is plagued by confusion about species identity and distribution. This study examines the taxonomic status of the *Rasbora* and *Nemacheilus* species in Java, Bali and Lombok islands through DNA barcodes, with the objective to resolve taxonomic confusion and identify trends in genetic diversity that can be further used for conservation matters. Several species delimitation methods based on DNA sequences were used and confirmed the status of most species, however several cases of taxonomic confusion and two new taxa are detected. Mitochondrial sequences argue that most species range distributions currently reported in the literature are inflated due to erroneous population assignments to the species level, and further highlight the sensitive conservation status of most *Rasbora* and *Nemacheilus* species on the islands of Java, Bali and Lombok.

**Keywords** Conservation genetics, taxonomy, Southeast Asia, cryptic diversity, population fragmentation

## Introduction

Biodiversity hotspots are characterized by high proportions of endemic species and high levels of anthropogenic threats (Myers et al. 2000). Identified to maximize conservation efforts in a world with finite human and funding resources for conservation matters, biodiversity hotspots have provided useful geographic proxies for conservation efforts. While those biodiversity hotspots have been delineated based on a limited set of well-known vertebrate taxa such as mammals, birds, amphibians and reptiles, the diversity and status of the world's most diverse vertebrate group, that is fishes, is still largely unknown (Myers et al. 2000; Lamoreux et al. 2006; Hoffman et al. 2010). With hundreds of new species described on a yearly basis, freshwater fishes suffer from an important taxonomic knowledge gaps that, combined with the taxonomic impediment (i.e. the rarefaction of taxonomists worldwide), currently plagues conservation efforts in most biodiversity hotspots (Winemiller et al. 2016; Garnett & Christidis 2017). This situation arguably accounts for their exclusion from most of the large-scale meta-analyses
conducted so far on global diversity patterns (Myers et al. 2000; Lamoreux et al. 2006; Hoffman et al. 2010).

In insular South-East Asia (SEA), the Sundaland hotspot exemplifies the stakes faced by conservation stakeholders due to antagonistic interests in the use of biological resources. Including the islands of Java, Sumatra and Borneo, Sundaland is currently among the largest hotspots in terms of number of species and endemics (Myers et al. 2000). Recent threat analyses, however rank it as one of the most threatened (Lamoreux et al. 2006; Hoffman et al. 2010). With nearly 900 species and 430 endemics, Sundaland accounts respectively for 74% and 48% of the total and endemic diversity of the approximately 1200 fish species cited from rivers of the Indonesian archipelago (Hubert et al. 2015). Within Sundaland, Java exhibits one the highest fish species density with 1.7 species per 1000 km² (213 species) together with Sumatra (460 species) and ahead of Kalimantan (Indonesian Borneo; 1.2 species per 1000 km² and 646 species). Hosting 130 million of people sharing 130000 km², Javanese aquatic ecosystems have faced a dramatic increase of anthropogenic threats during the last decade. The recent molecular inventory of the Javanese ichthyofauna evidenced large discrepancies between the checklist of Java freshwater fishes established from historical records (Hubert et al. 2015) and a modern reappraisal based on DNA sequences (Dahruddin et al. 2017), hence highlighting major gaps in the taxonomic knowledge of this ichthyofauna. Along the same line Hutama et al. (2017) evidenced high levels of cryptic diversity (i.e. morphologically unnoticed diversity) in widespread fish species of Java deriving from a late Pleistocene fragmentation of the populations associated with population bottlenecks. Considering that Sundaland is currently in a refugial state and that its emerged lands represent only a small fraction of its average surface during the Pleistocene (Woodruff 2010; Lohman et al. 2011), the state of Sundaland ichthyofauna urges for an acceleration of the ichthyological exploration of its freshwaters.

Initially designed to circumvent the taxonomic impediment by proposing a standard molecular framework for species identification through the use of the mitochondrial cytochrome oxidase I gene as an internal species tag, DNA barcoding opened new perspectives in the inventory of freshwater fishes (Hubert et al. 2008; Ward et al. 2009; Steinke & Hanner 2011). While large scale fish DNA barcoding campaigns have been tackled during the last decade (April et al. 2011; Hubert et al. 2012; Pereira et al. 2013; Geiger et al. 2014; Knebelsberger et al. 2015; Dahruddin et al. 2017; Durand et
al. 2017; Hubert et al. 2018; Machado et al. 2018), it becomes more and more evident that the pace of species description is surpassed by the astonishing underestimation of species diversity, often referring to cryptic diversity, and the complexity of fish biodiversity (Hubert et al. 2012; Jaafar et al. 2012; Kadarusman et al. 2012; Geiger et al. 2014; Winterbottom et al. 2014). We focus in the present study on the diversity and range distribution in South Sundaland of two Cypriniformes genera, namely *Rasbora* (Cyprinidae) and *Nemacheilus* (Nemacheilidae) that constitute emblematic endemic lineages in Java and Lesser Sunda Islands (Bali, Lombok) due to their occurrence in a large array of aquatic ecosystems and their high levels of endemism compared to other genera occurring in Java. Mostly described during the eighteenth and nineteenth centuries, *Rasbora* and *Nemacheilus* taxonomy and distribution is confusing in Java due to the lack of traceability of the taxonomic information often associated with old descriptions. Type localities are available for most of these species (Kottelat 2013), however range distribution are currently unknown (Froese & D 2014; Hubert et al. 2015; Eschmeyer et al. 2018), most *Rasbora* and *Nemacheilus* species being reported in Java and/or Bali without further details. With the aim to re-examine *Rasbora* and *Nemacheilus* diversity on the islands of Java, Bali and Lombok, we produced a DNA barcode reference library with the following objectives: (1) exploration of species biological boundaries through DNA-based species delimitation methods, (2) validation of species identity and taxonomy and precise range distribution by producing DNA barcodes from type localities or neighboring watersheds, (3) estimation of species genetic diversity and production of recommendations for conservation genetics purposes.

122

## 123 Materials and methods

124 Sampling and collection management

The authors previously conducted a large-scale DNA barcoding campaign across 95 sites in Java and Bali Island between November 2012 and May 2015 (Dahruddin et al. 2017). During this initial inventory, a total of 3310 specimens, including 162 species belonging to 110 genera and 53 families were collected. This was complemented by an additional campaign in Lombok island on March 2015 resulting in the sampling of an additional set of 367 specimens belonging to 54 species and 44 genera sampled across 12 sites. With the objective to produce a DNA barcode reference library for the Java and Bali
132 ichthyofauna, a total of 24 specimens for 4 species of *Rasbora* and 15 specimens for 2
133 species of *Nemacheilus* were previously sequenced (Dahruddin et al. 2017). Considering
134 the objectives of the present study, an additional set of 84 specimens of *Nemacheilus* and
135 118 specimens of *Rasbora* were selected at all the sites these genera were sampled
can during the initial campaign for further sequencing (Fig. 1). Thus, a total of 99 specimens
137 belonging to 2 species of *Nemacheilus* and 142 specimens belonging to 4 species of
138 *Rasbora* were analyzed in the present study (Table S1).

139 Specimens were captured using various gears including electrofishing, seine nets,
cast nets and gill nets across sites encompassing the diversity of freshwater lentic and
140 lotic habitats. Specimens were identified following available monographs (Kottelat et al.
141 1993), and species names were further validated based on several online catalogues
142 (Froese & D 2014; Eschmeyer et al. 2018). Specimens were photographed and
143 individually labeled, and voucher specimens were preserved in a 5% formalin solution.
144 A fin clip or a muscle biopsy was taken for each specimen and fixed in a 96% ethanol
145 solution for genetic analyses. Both tissues and voucher specimens were deposited in the
146 national collections at the Muzeum Zoologicum Bogoriense (MZB) in the Research
147 Centre for Biology (RCB) from the Indonesian Institute of Sciences (LIPI).
148
149

150

Sequencing and international repositories

151 Genomic DNA was extracted using a Qiagen DNeasy 96 tissue extraction kit
152 following the manufacturer's specifications. A 651-bp segment from the 5' region of the
153 cytochrome oxidase I gene (COI) was amplified using primer cocktails
154 C_FishF1t1/C_FishR1t1 including M13 tails (Ivanova et al. 2007). PCR amplifications
155 were done on a Veriti 96-well Fast (ABI-AppliedBiosystems) thermocycler with a final
156 volume of 10.0µl containing 5.0µl Buffer 2X, 3.3µl ultrapure water, 1.0µl each primer
157 (10µM), 0.2µl enzyme Phire® Hot Start II DNA polymerase (5U) and 0.5µl of DNA
158 template (~50 ng). Amplifications were conducted as follow: initial denaturation at 98°C
159 for 5 min followed by 30 cycles denaturation at 98°C for 5s, annealing at 56°C for 20s
160 and extension at 72°C for 30s, followed by a final extension step at 72°C for 5 min. The
161 PCR products were purified with ExoSap-IT® (USB Corporation, Cleveland, OH, USA)
162 and sequenced in both directions. Sequencing reactions were performed using the
163 "BigDye® Terminator v3.1 Cycle Sequencing Ready Reaction" and sequencing was
164 performed on the automatic sequencer ABI 3130 DNA Analyzer (Applied Biosystems).
The sequences and collateral information have been deposited in BOLD (Ratnasingham & Hebert 2007) and are available in the projects BIFH, BIFHB, BIFI & BIFB. DNA sequences were submitted to GenBank (accession numbers are accessible directly at the individual records in BOLD).

## Species delimitation and genetic diversity

A maximum likelihood (ML) tree was first reconstructed using phyml 3.0.1 (Guindon 2003) based on the most likely substitution model selected by JMODELTEST 2.1.7 (Darriba et al. 2012). An ultrametric and fully resolved tree was reconstructed using the Bayesian approach implemented in BEAST 2.4.8 (Bouckaert et al. 2014). Two markov chain of 50 million each were ran independently using Yule pure birth model tree prior and an uncorrelated relaxed lognormal clock model for both *Rasbora* and *Nemacheilus* data sets. The ML tree was converted into an ultrametric tree using a relaxed clock model of the chronos function in the R package Ape 4.1 (Paradis 2004) and further used to initiate tree searches for the Bayesian analyses. Calibrations of ML and Bayesian analyses were established following Hutama et al. (2017). Age intervals for the Most Recent Common Ancestor (MRCA) of *Rasbora* spp and *Nemacheilus* spp were estimated based on the canonical 1.2% (+/- 0.5%) of genetic distance per million years for the fish COI gene (Bermingham et al. 1997). The average genetic distances between species pairs involving a direct ancestry with the MRCAs of *Rasbora* and *Nemacheilus* were calculated using MEGA 6 (Tamura et al. 2013) and used to estimate the age interval of the MRCAs. An additional calibration was added in the *Rasbora* tree including *Rasbora baliensis*, *R. lateristriata* and *R. aprotaenia* and also in *Nemacheilus* tree for the MRCA of *N. chrysolaimos* haplotypes following the same methodology. Trees were sampled every 10,000 states after an initial burning period of 10 million and both runs were combined using LogCombiner 2.4.8 (Bouckaert et al. 2014). The maximum credibility tree was constructed using TreeAnnotator 2.4.7 (Bouckaert et al. 2014).

Several alternative methods have been proposed for delimitating molecular lineages (Pons et al. 2006; Puillandre et al. 2012; Ratnasingham & Hebert 2013b; Zhang et al. 2013; Hubert & Hanner 2015). These methods rely on different approaches and assumptions but they all have in common the detection of transitions between mutation/drift (within species) and speciation/extinction (between species) dynamics (Hubert & Hanner 2015). Each of these methods is prone to pitfalls, particularly
regarding singletons (i.e. delimitated lineages represented by a single sequence) and
combining different approaches is increasingly used to circumvent potential pitfalls
arising from, for instance, uneven sampling among species (Kekkonen & Hebert 2014;
Kekkonen et al. 2015; Blair & Bryson 2017). Here, four sequence-based methods of
species delimitation were used to delimitate species, and a final delimitation scheme
was established based on a 50 percent consensus among methods in order to produce a
robust delimitation scheme. For the sake of clarity, species identified based on
morphological characters are referred to as species while species delimitated by DNA
sequences are referred to as Operational Taxonomic Units (OTU), defined as diagnosable
molecular lineages (Avise 1989; Moritz 1994; Vogler & DeSalle 1994; Hutama et al.
2017). OTUs were delimitated using the following algorithms: (1) Refined Single
Linkage (RESL) as implemented in BOLD and used to produce Barcode Index Numbers
(BIN)(Ratnasingham & Hebert 2013a), (2) Automatic Barcode Gap Discovery
(ABGD)(Puillandre et al. 2012), (3) Poisson Tree Process (PTP) in its multiple rates
version (mPTP) as implemented in the stand-alone software mptp_0.2.3 (Zhang et al.
2013; Kapli et al. 2017), and (4) General Mixed Yule-Coalescent (GMYC) in its single rate
version (sGMYC) as implemented in the R package Splits 1.0-19 (Fujisawa &
Barraclough 2013). RESL and ABGD used the DNA alignments as inputs while the ML
tree was used for mPTP. Two delimitation schemes were collected for sGMYC: (1) a
scheme based on the Maximum credibility tree from the Bayesian analysis as input
(sGMYC), (2) a consensus scheme with OTUs selected if present in more than 50% of the
10 replicates of sGMYC based on 10 Bayesian trees sampled along the Markov chain
(sGMYC*).

We quantified the match among methods and their relative power using the match ratio, the Relative Taxonomic Index of Congruence index ($R_{tax}$) and the Taxonomic Index of Congruence ($C_{tax}$) following Blair and Bryson (2017). The match ratio is a measure of concordance among methods and is defined as twice the number of matches divided by the sum of the number of delimitated OTUs and the number of morphological species (Arhens et al. 2016). The $R_{tax}$ index quantifies the relative power of a method to infer all estimated speciation events and is defined as the number of speciation events identified by a method divided by the total number of speciation events identified by the different methods (Miralles & Vences 2013). The $C_{tax}$ index is a measure of congruence in species assignments between two methods and is calculated by dividing the number
of speciation events inferred jointly by the two methods by the total number of
speciation events inferred. Considering the number of comparisons involved, an average
$C_{tax}$ index was calculated for each method.

For each species, Kimura 2-parameter (K2P) pairwise genetic distances were
calculated using the R package Ape 4.1 (Paradis 2004). Maximum intraspecific and
nearest neighbor genetic distances were calculated from the matrix of pairwise K2P
genetic distances using the R package Spider 1.5 (Brown et al. 2012). Haplotype
diversity (h) and nucleotide diversity (π) were calculated for each species using the R
package Pegas 0.1 (Paradis 2010).

240

## 241 Results

99 and 142 sequences were successfully obtained for *Nemacheilus* and *Rasbora* respectively. All the sequences were above 500 bp of length and no stop codons were detected, suggesting that the sequences collected represent functional coding regions. The maximum credibility tree of *Rasbora* spp identified a group of closely related species including *R. aprotaenia*, *R. lateristriata* and *R. baliensis* as well as two unknown taxa labeled as *R. sp1* and *R. sp2* (Fig. 2). The age of the MRCA of this clade of closely related species is inferred to trace back around 3 million years ago (Ma), and the split between *Rasbora argyrotaenia* and the remaining *Rasbora* is inferred to happen around 11 Ma. The age of *Rasbora* species MRCAs ranged between 0.5 Ma for *R. baliensis* and 1 Ma for *R. sp1*. The maximum credibility tree clearly separated the two *Nemacheilus* species genealogy into two distinct clades with a MRCA dated around 10 Ma (Fig. 3). The MRCA of *N. chrysolaimos* and *N. fasciatus* genealogies are dated around 1.5 and 0.5 Ma respectively.

Delimitation methods largely converged in identifying 8 OTUs within the 6 species of *Rasbora* recognized here. Of the two partitioning schemes obtained with sGMYC, only the consensus partitioning scheme derived from 10 replicates (sGMYC*) is consistent with other methods in *Rasbora* (Fig. 1), with a number of OTUs ranging from 7 to 9 across sampled trees. Applying sGMYC to the Bayesian maximum credibility tree resulted in an inflated number of OTUs as 51 lineages were delineated (Table 1). Two OTUs were detected within *R. lateristriata* and *R. sp1*. The match ratio was similar among methods excepting sGMYC and the highest resolution power was observed for sGMYC with a *Rtax* of 1 (Table 1). The highest taxonomy congruence was observed for
BIN, ABGD, mPTP and sGMYC* with a *C*<sub>tax</sub> of 0.784 (Table 1). Delimitation methods produced concordant delimitation schemes within *Nemacheilus*, as all methods, excepting sGMYC, delineated one OTU for each of the two species (Fig. 3). As observed for *Rabora*, sGMYC inflated the number OTUs with 4 OTUs delimitated within *N. chrysolaimos* (Table 2). The *match ratio* and the taxonomic concordance (*C*<sub>tax</sub>) were the highest for all methods excepting sGMYC (Table 2). The resolution power was estimated to be the highest for sGMYC as revealed by *R*<sub>tax</sub> (Table 2).

Maximum intraspecific K2P distances ranged between 0.62 in *R. argyrotaenia* and 2.51 in *R. sp1* (Table 1), and the minimum nearest neighbor K2P distance ranged between 2.68 in *R. aprotaenia* and 9.67 in *R. argyrotaenia*. Haplotype diversity was globally high for all species with a haplotype diversity ranging from 0.795 in *R. aprotaenia* to 0.505 in *R. baliensis*. Nucleotide diversity was low for all species, with the lowest values ranging from 0.001 in *R. argyrotaenia* to 0.004 in *R. aprotaenia*, excepting for *R. lateristriata* and *R. sp1*, the only two species with two OTUs (Table 1). Maximum intraspecific K2P distances were 0.47 and 1.72 for *N. fasciatus* and *N. chrysolaimos*, respectively and the K2P distance between the two species was 14.78 (Table 2). Genetic diversity is markedly different between the two *Nemacheilus* species with *N. fasciatus* exhibiting a low genetic diversity with a haplotype diversity of 0.168 and a nucleotide diversity of 0.001 while *N. chrysolaimos* has a haplotype diversity of 0.828 and a nucleotide diversity of 0.007 (Table 2).

284

## Discussion

### Taxonomy of *Rasbora* species in Java, Bali and Lombok

Species boundaries of the four *Rasbora* species were successfully recovered by all, except sGMYC, species delimitation methods. This result has several implications regarding the taxonomy of the *Rasbora* genus in Java. The morphological characters described by Kottelat (1993) were not all operational as different and non-standardized meristic features were described for each species. Coloration patterns were used to propose an initial set of morphological identifications that resulted in the acknowledgement of four species based on the following key: (1) *R. aprotaenia* is distinguished by two dark spots along the lateral line being connected by a thin and diffuse dark line, the first below the origin of the dorsal fin, the second on the caudal peduncle and a dark spot along the proximal margin of the anal fin (Fig. 4). (2) *R.*
297 *argyrotaenia* can be further separated in having a continuous dark line that cover nearly 298 entirely the lateral line and a dark line that underlines the entire proximal margin of the 299 anal fin. (3) *R. laterstriata* and *R. baliensis* are further distinguished by the number of 300 scale on the lateral line with 26-28 in *R. baliensis* and 29-33 in *R. laterstriata* (Kottelat et 301 al. 1993). The concordance between the DNA-based and coloration- and meristic-based 302 delimitation schemes for the four known *Rasbora* species confirms their biological 303 species status. In addition, the examination of range distributions and known type 304 localities further confirm this concordance with type localities being contained within 305 the observed distribution range for *R. argyrotaenia*, *R. aprotenia* and *R. baliensis* (Figs. 306 5a, 5b, 5d).

307 *R. laterstriata* has been initially described based on a series of specimens 308 collected throughout the Western part of Java (Kottelat 2013) at type localities that are 309 not included in the range distribution observed here (Fig. 5c). Most of the type localities 310 belong to the watershed draining the largest urban areas in Java (i.e. Jakarta, Bogor, 311 Bandung), which are highly impacted by anthropogenic activities. Previous observations 312 highlighted that some localities in this part of Java were dominated by invasive species 313 including *Xyphophorus* spp., *Oreochromis niloticus*, *Clarias gariepinus* and *Poecilia* spp. 314 (Dahruddin et al. 2017), and very few of the sites visited in the western part of Java 315 resulted in the capture of *Rasbora* specimens (Jawa Barat, Table S1). Also it is possible 316 that we failed to capture *R. laterstriata* while the species was present, this result 317 suggests that it has become rare, at least in this part of Java. The concordance between 318 morphology-based and sequence-based species delimitation, however seems to confirm 319 its validity.

320 *Rasbora baliensis* was reported as a species endemic to the crater lakes in Bali 321 while *R. laterstriata* replaced it in the rivers of Bali (Kottelat et al. 1993; Kottelat 2013). 322 Our study shows that *R. baliensis* has a range distribution surprisingly wider than 323 expected as its presence is detected until East Java and Lombok (Fig. 5d). The confusion 324 that reigns over the known range distribution of *R. baliensis* and *R. laterstriata* is 325 surprising considering that they show non-overlapping numbers of scale at the lateral 326 line, a character that has been previously overlooked. The otherwise morphological 327 similarity between the two species is likely to account for the reported occurrence of *R. 328 laterstriata* in most Lesser Sunda Islands including Bali, Lombok and Sumbawa 329 (Kottelat et al. 1993). The disjunctive range distribution of both species, as well as the
reciprocal monophyly that was captured by all DNA-based delimitation methods, argue that *R. baliensis* is a valid taxon. Furthermore, the presence of *R. baliensis* in east Java and Lombok is reported here for the first time while we show that *R. lateristriata* is restricted to central Java (Fig. 5d).

Finally, two new taxa are discovered here, *Rasbora* sp1 and *Rasbora* sp2 (Fig. 2; Fig. 4). The COI tree suggests that both taxa are closely related to *R. aprotaenia*. The coloration pattern of *R. sp1* is markedly different from *R. aprotaenia*, however as *R. sp1* exhibit a thin dark line, instead of a dark spot in *R. aprotaenia*, at the proximal margin of the anal fin and a thin and a diffuse dark line along the lateral line (Fig. 4e). *Rasbora* sp2 is much more similar to *R. aprotaenia* in terms of coloration pattern, and the fact that a single specimen was captured warrants further studies to enable its formal description. The diversification of this clade of closely related species is inferred to happen during the last 2.5 Ma in a restricted area of the western part of Java, a pattern previously described for several species largely distributed in Java such as *Barbodes binotatus*, *Channa gachua* and *Glyptothorax platypogon* (Hutama et al. 2017).

## Taxonomy of *Nemacheilus* species in Java

The DNA-based delimitation schemes are consistent with the presence of two species in Java, namely *Nemacheilus fasciatus*, reported to occur in Java and Sumatra, and *Nemacheilus chrysolaimos*, an endemic species of Java (Kottelat et al. 1993; Kottelat 2013). The examination of the type locality of *N. fasciatus* provided a conflicting result with the initial identification performed based on the diagnostic morphological characters described in Kottelat (1993). The type locality of *N. chrysolaimos* is unknown and the type locality of *N. fasciatus* is located in the western part of Java (Fig. 6). Following Kottelat's monograph (1993), the Western species is *Nemacheilus chrysolaimos*. According to our study, however the *Nemacheilus* species of western Java is *N. fasciatus*, *N. chrysolaimos* being largely distributed in Central and Eastern Java (Fig. 6). A reexamination of the diagnostic morphological characters proposed to differentiate both *Nemacheilus* species in Java at the light of the present results indicates that *N. fasciatus* is the species that is distinguished in having a winged flap on the margin of the anterior naris (Figs. 7c, 7d), contrasting with an anterior naris valve pierced at the tip of a tube in *N. chrysolaimos* (Figs. 7a, 7b). The coloration patterns of each species are also distinct with *N. chrysolaimos* presenting 14-18 dark blotches along lateral line with 11-
12 dark saddles across the back contrasting with *N. fasciatus* exhibiting 9-18 dark bars of irregular shape (Kottelat et al. 1993). The identity of both species was further confirmed by the examination of the type specimens of both species (*N. chrysolaimos*, B2972, B3961; *N. fasciatus*, B2798) deposited at the MNHN Paris, as well as the original illustration of *N. chrysolaimos* depicting a coloration pattern consisting of irregular saddles, consistent with the coloration pattern of the specimens assigned to *N. chrysolaimos* in the present study.

## Species distribution and conservation genetics

All the species under study here were previously reported as endemic species of the islands of Java, Bali and Lombok. The present study highlights that most of the species examined here have range distribution much more restricted than previously acknowledged. Excepting *N. chrysolaimos* and *R. argyrotaenia* detected in east and central Java, most species are confined to a restricted set of watersheds. A clear separation is observed between the eastern versus the central and east Java lineages, a pattern previously reported among cryptic lineages within widespread species in Java such as *Barbodes binotatus*, *Glyptothorax platypogon* and *Channa gachua* (Dahruddin et al. 2017; Hutama et al. 2017). This biogeographic transition between East and West watersheds in Java is related to the ontogeny of Java, resulting from the merging of two volcanic arches, one is the west and a second in the east (including East Java and Bali), that emerged between 10 and 5 Ma and further aggregated during the last 5 Million years (Lohman et al. 2011). This scenario is reflected in *Rasbora* with the range distribution of *R. baliensis* encompassing the eastern part of Java, Bali and Lombok. A similar trans-distribution across the Bali strait was previously observed in *Barbodes binotatus* and *Channa gachua* (Hutama et al. 2017). Divergence levels between the western most species and the central/east species are surprisingly higher than previously observed in Java with a divergence between *N. fasciatus* and *N. chrysolaimus* inferred at around 10 Ma (1.01-17.90 Ma, 95% HPD) and the divergence of *R. argyrotaenia* from other *Rasbora* inferred at around 11.5 Ma (10.61-12.55 Ma, 95% HPD). These age estimates are much older than reported for other widespread species in Java with species MRCA estimated to occur around 3 Ma for several species (Hutama et al. 2017). The age of the MRCA of sampled species of *Nemacheilus* and *Rasbora* also trace back beyond the ontogeny of Java, suggesting that the evolutionary history of these
Javanese species started prior to the rise of this island, during the initial settlement of Sundaland (Lohman et al. 2011). These old splits particularly contrast in *Rasbora* with divergence age estimates for other *Rasbora* species that do not exceed 3.5 Ma, an age consistent with previous age estimates of the MRCA of the coalescent trees of several widespread species in Java (Hutama et al. 2017). Interestingly, *N. fasciatus* and *R. argyrotaenia* are both cited from Sumatra suggesting a recent colonization of Java from Sumatra for those species. This scenario was recently suggested for *Rasbora* based on a phylogenetic assessment of Java species based on mitochondrial genomes (Kusuma et al. 2016), and highlights the different origins of the western and eastern species of *Nemacheilus* and *Rasbora*. The *Rasbora* tree topology, supporting close relationships between central and east Java species, and age estimates, below 3.5 Ma, suggest that most *Rasbora* species diversified through in-situ speciation in Java, a hypothesis previously suggested (Kusuma et al. 2016).

The general trend of highly restricted range distribution, related to a hypothesized origin though in-situ diversification in Java and Bali for *Rasbora*, highlights the sensitive conservation status of most endemic species in Java. The distribution patterns observed here are highly fragmented with little overlap between range distributions of sister-species, a pattern that was expected considering the volcanic origin of Java, Bali and Lombok islands during the Quaternary (Lohman et al. 2011). Genetic diversity patterns are consistent with these observations as nucleotidic diversity is generally low for *Rasbora* and *Nemacheilus* species, a molecular signature also consistent with a recent origin through in-situ diversification. Cryptic diversity is also reported here for *R. lateristriata* and *R. sp1*, with cryptic lineages diverging around 1 Ma for both species, further accentuating the fragmented status of the populations of these species. As such, most *Rasbora* species should be considered as complexes of small populations, highly sensitive to further reductions in population size that may eventually lead to a vortex of extinction (Gilpin & Soulé 1986; Fagan & Holmes 2006).

Java is the most densely populated island in Sundaland and anthropogenic activities have an alarming impact on the rivers of this region (Spracklen et al. 2015; Breckwoldt et al. 2016; Hayati et al. 2017; Garg et al. 2018). Severe reductions of aquatic habitat size during the last two decades have been reported with significant impacts of water pollution generated by industrial wastes and agricultural run-offs, but also the introduction of exotic species (Eidman 1989; Dahruddin et al. 2017). Considering the
very narrow range distributions observed here for most endemic species and the concerning environmental context of Java, this study stresses that conservation efforts even at a small spatial scale could have significant impacts in the conservation of these endemic species. Translocations of livestock are common in Indonesia and are often used as a management measure (Dahruddin et al. 2017). The presence of cryptic diversity, newly discovered, yet undescribed, taxa and their close morphological affinities make accurate species identifications difficult. In this context, the introduction of multiple species through translocation programs due to species misidentifications are likely if not guided by DNA barcoding. Considering the young age of most of the species examined here and their allopatric distributions, the effectiveness of reproductive isolation mechanisms after secondary contact is questionable and the occurrence of hybridization cannot be discarded. Both genera have become scarce in the Western part of Java. Combined with the low genetic diversity at the nucleotide level, restoration program through genetic rescue (i.e. increasing population fitness through the repletion of genetic diversity by immigration) are probably required (Whiteley et al., 2014). If such translocation programs were to be implemented in the future, we advocate for short spatial scale translocations in order to avoid secondary contact among closely related species and identify immigrants, to the species level using DNA barcodes.

447

## Conclusion

As in previous molecular studies in the area, the present study highlights gaps of knowledge in the taxonomy and distribution of the freshwater fishes of Java and Bali, and further highlights the complexity of diversity patterns in this part of Sundaland. This assessment of *Rasbora* and *Nemacheilus* diversity through DNA barcodes sheds light on the species biological status and distribution in South Sundaland and warrants further studies. In particular, the two new taxa discovered here need additional sampling efforts to be accurately described. The present study highlights the sensitive status of most species owing to their restricted range and low genetic diversity.

457

## Acknowledgements

The authors wish to thank Siti Nuramaliati Prijono, Bambang Sunarko, Witjaksono, Mohammad Irham, Marlina Adriyani, Ruliyana Susanti, Rosichon Ubaidillah, Hari Sutrisno and Muhamad Syamsul Arifin Zein at Research Centre for Biology (RCB-
462 LIPI); Jean-Paul Toutain, Robert Arfi, Valérie Verdier and Jean-François Agnès from the
463 'Institut de Recherche pour le Développement'; Joel Le Bail and Nicolas Gascoin at the
464 French embassy in Jakarta for their continuous support. We are thankful Sumanta at IRD
465 Jakarta for his help during the field sampling. Part of the present study was funded by
466 the Institut de Recherche pour le Développement (UMR226 ISE-M and IRD through
467 incentive funds), the MNHN (UMR BOREA), the RCB-LIPI, the French Ichthyological
468 Society (SFI), the Foundation de France and the French embassy in Jakarta. The
469 Indonesian Ministry of Research and Technology approved this study and field sampling
470 was conducted according to the research permits 097/SIP/FRP/SM/IV/2014 for
471 Philippe Keith, 60/EXT/SIP/FRP/SM/XI/2014 for Frédéric Busson and
472 41/EXT/SIP/FRP/SM/VIII/2014 for Nicolas Hubert. Sequence analysis was aided by
473 funding from the government of Canada through Genome Canada and the Ontario
474 Genomics Institute in support of the International Barcode of Life project. We thank Paul
475 Hebert, Robert Hanner and Evgeny Zakharov as well as BOLD and CCDB staffs at the
476 University of Guelph for their valuable support. Finally, we thank Anti Vasemägi and the
477 three anonymous reviewers for providing constructive comments of earlier versions of
478 the manuscript. This publication has ISEM number 2018-279-SUD.

479

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653
654
**Fig. 1** Collection sites for the 241 samples analyzed in the present study following the sampling campaign detailed in Dahruddin et al. 2017 and new sampling events in Lombok island. **A** Collection sites of *Rasbora* specimens. **B** Collection sites of *Nemacheilus* specimens. White dots correspond to sites where *Rasbora* or *Nemacheilus* specimens were collected. Black dots represent visited sites where no *Rasbora* or *Nemacheilus* specimens were observed. Each dot may represent several collection sites.

<figure><img src="image_2.png" /></figure>

661

662
Fig. 2 Bayesian Maximum Credibility Tree of *Rasbora* DNA barcodes including 95% HPD interval for node age estimates and sequence clustering results according to the 5 species delimitation methods implemented.

<figure><img src="image_3.png" /></figure>
**Fig. 3** Bayesian Maximum Credibility Tree of *Nemacheilus* DNA barcodes including 95% HPD interval for node age estimates and sequence clustering according to the 5 species delimitation methods implemented.

<figure><img src="image_4.png" /></figure>
Fig. 4 Selected specimen photographs of each of the 6 *Rasbora* species collected and recognized in the present study. **a Rasbora aprotaenia** (specimen BIF1501; SL=47mm; Ci Siih, Banten, Java). **b Rasbora argyrotaenia** (specimen BIF976; SL=33mm; Cilacap, Central Java). **c Rasbora baliensis** (specimen BIF2351; SL=72mm; Jembrana, West Bali). **d Rasbora lateristriata** (specimen BIF3619; SL=87mm; Kali Dauwan, Mojokerto, East Java). **e Rasbora** sp1 (specimen BIF864; SL=61mm; Kali Pelus, Purwokerto, Central Java). **f Rasbora** sp2 (specimen BIF155; SL=43mm; Ci Heulang, Sukabumi, West Java).

<figure><img src="image_5.png" /></figure>
Fig. 5 Species range distribution of the 6 *Rasbora* species recognized in the present study. Colored dots represent collection sites. White circles represent type localities.
a. *Rasbora aprotaenia*
b. *Rasbora argyrotaenia*
c. *Rasbora lateristriata*
d. *Rasbora baliensis*
e. *Rasbora* sp1.
f. *Rasbora* sp2.

<figure><img src="image_6.png" /></figure>

687

688

689
Fig. 6 Species range distribution of the 2 *Nemacheilus* species recognized in the present study. Colored dots represent collection sites. White circles represent type localities.

**a** *Nemacheilus chrysolaemos*. **b** *Nemacheilus fasciatus*.

<figure><img src="image_7.png" /></figure>

693

694

695
Fig. 7 Selected specimen photographs of each of the 2 *Nemacheilus* species collected and recognized in the present study. **a** *Nemacheilus fasciatus* (specimen BIF495; SL=42mm; Ci Asem, Purwakarta, West Java). **b** *Nemacheilus fasciatus* (specimen BIF163; SL=45mm; Ci Heulang, Central Java). **c** *Nemacheilus chrysolaimos* (specimen BIF2032; SL=58mm; Ngerjo, Blitar, East Java). **d** *Nemacheilus chrysolaimos* (specimen BIF2074; SL=55mm; Bicoro, Lumajang, East Java).

<figure><img src="image_8.png" /></figure>
703

704

**Table 1** Summary statistics of *Rasbora* species genetic diversity and species delimitation schemes. *n*, number of individual sequences analyzed. Max. K2P distance, maximum K2P genetic distance (percent). Min. Nearest Neighbor K2P distance (percent), minimum K2P genetic distance to the nearest phylogenetic neighbor (percent) in the present data set. *h*, haplotypic diversity. *π*, nucleotidic diversity. match ratio, twice the number of match divided by the sum of the number of delimitated OTU and the number of morphological species. *R<sub>tax</sub>*, number of speciation events identified by a method divided by the total number of speciation events identified by the different methods. *C<sub>tax</sub>*, average number of speciation events inferred jointly by two methods divided by the total number of speciation events inferred.

705

706

707

708

709

710

<table><thead><tr><th>Taxon</th><th>n</th><th>Max. K2P distance</th><th>Min. Nearest Neighbor K2P distance</th><th>h</th><th>π</th><th>BIN</th><th>ABGD</th><th>bPTP</th><th>sGMYC</th><th>sGMYC*</th></tr></thead><tbody><tr><td><i>R.</i></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><i>argyrotaenia</i></td><td>20</td><td>0.62</td><td></td><td>9.67</td><td>0.742</td><td>0.001</td><td>1</td><td>1</td><td>1</td><td>8</td><td>1</td></tr><tr><td><i>R. aprotaenia</i></td><td>27</td><td>0.93</td><td></td><td>2.68</td><td>0.795</td><td>0.004</td><td>1</td><td>1</td><td>1</td><td>10</td><td>1</td></tr><tr><td><i>R. lateristriata</i></td><td>20</td><td>2.08</td><td></td><td>3.04</td><td>0.753</td><td>0.009</td><td>2</td><td>2</td><td>2</td><td>8</td><td>2</td></tr><tr><td><i>R. baliensis</i></td><td>54</td><td>0.77</td><td></td><td>3.04</td><td>0.505</td><td>0.002</td><td>1</td><td>1</td><td>1</td><td>16</td><td>1</td></tr><tr><td><i>R. sp1</i></td><td>20</td><td>2.51</td><td></td><td>3.33</td><td>0.658</td><td>0.012</td><td>2</td><td>2</td><td>2</td><td>8</td><td>2</td></tr><tr><td><i>R. sp2</i></td><td>1</td><td>NA</td><td></td><td>NA</td><td>NA</td><td>NA</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>Total</td><td>142</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>8</td><td>8</td><td>8</td><td>51</td><td>8</td></tr><tr><td>match ratio</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>0.571</td><td>0.571</td><td>0.571</td><td>0.035</td><td>0.571</td></tr><tr><td>Rtax</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>0.16</td><td>0.16</td><td>0.16</td><td>1</td><td>0.16</td></tr><tr><td>Mean Ctax</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>0.784</td><td>0.784</td><td>0.784</td><td>0.04</td><td>0.784</td></tr></tbody></table>

711
712

713

714

715

716

717

718

719

**Table 2** Summary statistics of *Nemacheilus* species genetic diversity and species delimitation schemes. *n*, number of individual sequences analyzed. Max. K2P distance, maximum K2P genetic distance (percent). Min. Nearest Neighbor K2P distance (percent), minimum K2P genetic distance to the nearest phylogenetic neighbor (percent) in the present data set. *h*, haplotypic diversity. *π*, nucleotidic diversity. match ratio, twice the number of match divided by the sum of the number of delimitated OTU and the number of morphological species. *R*<sub>tax</sub>, number of speciation events identified by a method divided by the total number of speciation events identified by the different methods. *C*<sub>tax</sub>, average number of speciation events inferred jointly by two methods divided by the total number of speciation events inferred.

<table><thead><tr><th>Taxon</th><th>n</th><th>Max. K2P distance</th><th>Min. Nearest Neighbour K2P distance</th><th>h</th><th>π</th><th>BIN</th><th>ABGD</th><th>bPTP</th><th>sGMYC</th><th>sGMYC*</th></tr></thead><tbody><tr><td>N. fasciatus</td><td>14</td><td>0.47</td><td></td><td>14.78</td><td>0.168</td><td>0.001</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>N. chrysolaimos</td><td>85</td><td>1.72</td><td></td><td>14.78</td><td>0.828</td><td>0.007</td><td>1</td><td>1</td><td>1</td><td>4</td></tr><tr><td>Total</td><td>99</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>2</td><td>2</td><td>2</td><td>5</td></tr><tr><td>match ratio</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>1</td><td>1</td><td>1</td><td>0.286</td></tr><tr><td>Rtax</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>0.25</td><td>0.25</td><td>0.25</td><td>1</td></tr><tr><td>Mean Ctax</td><td>-</td><td>-</td><td></td><td>-</td><td>-</td><td>-</td><td>0.813</td><td>0.813</td><td>0.813</td><td>0.25</td></tr></tbody></table>

720

721

722
723 Supplementary materials

724 Fig. S1 Illustration of *Nemacheilus chrysolaimos* in its original description.

<figure><img src="image_9.png" /></figure>

LOCHE aux barbes d'or
Outilart p. it.

COBITIS Chrysolaimos nob.
Arundinaceae

725

726 **Table S1 Collection information**