Molecular Phylogeny of Catfishes (Teleostei: Siluriformes...

Molecular Phylogeny of Catfishes (Teleostei: Siluriformes) in the Philippines Using the Mitochondrial Genes COI,

Cyt b, 16S rRNA, and the Nuclear Genes Rag1 and Rag2

Philippine Journal of Science143 (2): 187-198, December 2014ISSN 0031 - 7683Date Received: 02 February 2014

Shiny Cathlynne S. Yu and Jonas P. Quilang*

Molecular Population Genetics Laboratory, Institute of Biology,College of Science, University of the Philippines,

1101Diliman, Quezon City, Philippines

In this study, three mitochondrial genes, namely, cytochrome c oxidase subunit I (COI), cytochrome b (cyt b), and 16S rRNA, and two nuclear genes, namely, recombination activating gene 1 (rag1) and recombination activating gene 2 (rag2) were used to determine the phylogenetic relationships of seven native and four introduced catfishes in the Philippines belonging to five families. All genetic trees constructed using the methods Maximum-Likelihood (ML) and Bayesian inference (BI) of concatenated sequences of the five genes support the monophyly of catfishes in each of the five families. ML and BI generated a topology (Loricariidae + (Clariidae + (Ariidae + (Pangasiidae + Plotosidae)))). Loricariidae is separated from the monophyletic clade of Ariidae, Pangasiidae, Clariidae and Plotosidae. One specimen each of Arius manillensis and A. dispar (Ariidae) shared the same unique concatenated sequence, while two specimens of Pterygoplichthys pardalis shared a unique concatenated sequence with one specimen of P. disjunctivus (Loricariidae). It is possible that the two Arius species and the two Pterygoplichthys species are synonymous. Future studies may use cytogenomic markers to establish if the species of these latter genera are valid. Future studies may also use a combination of molecular and morphological data in inferring the phylogenetic relationships of catfishes.

Key Words: catfishes, mitochondrial gene, nuclear gene, phylogeny, Siluriformes

*Corresponding author: [email protected]

INTRODUCTIONCatfishes (Order Siluriformes) are a diverse group of ray-finned fishes (Nelson 2006) that are distributed in all continents (Diogo 2004) and comprise more than 3,088 valid species distributed among 477 genera and 36 families (Ferraris 2007). Catfishes are valued as popular sport fish, food items, and tropical aquarium fish (Nelson 2006). Two introduced species, namely, Clarias gariepinus and Pangasianodon hypophthalmus are used in aquaculture, while two other introduced species, namely,

Pterygoplichthys disjunctivus and Pterygoplichthys pardalis are causing environmental problems.

Catfishes are primarily freshwater fishes with only two marine families: Plotosidae, which is distributed in the Indo-West Pacific and Ariidae, which is found worldwide in tropical to warm temperate zones (Kailola 2004). In the Philippines, Froese & Pauly (2013) in FishBase list 31 species of catfishes belonging to eight families, namely, Ariidae, Clariidae, Callichthyidae, Loricariidae, Ictaluridae, Pangasiidae, Plotosidae, and Siluridae. However, Corydoras aeneus, the single species of Callichthyidae, is questionable as it was only reported

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as a specimen in a fish living museum (Froese & Pauly 2013). Likewise, the occurrence of Ictalurus punctatus, the single species of Ictaluridae, is questionable as this species was introduced in 1974 but it could not thrive in the natural conditions of the Philippines (Juliano et al. 1989). Only 15 catfish species do not have contradicting reports and are certain to occur in the Philippines. These include Arius dispar, Arius manillensis, Plicofollis magatensis, Plicofollis nella, Clarias batrachus, Clarias gariepinus, Clarias macrocephalus, Clarias nieuhofii, Pterygoplichthys disjunctivus, Pterygoplichthys pardalis, Pangasianodon hypophthalmus, Paraplotosus albilabris, Plotosus canius, Plotosus lineatus, and Pterocryptis taytayensis. Eleven species of the catfishes reported in the Philippines have been DNA barcoded using the cytochrome c oxidase subunit I (COI) gene (Quilang & Yu 2013). These species include the endemic Arius manillensis Valenciennes 1840 and Plicofollis magatensis (Herre 1926), the native Arius dispar Herre 1926, Clarias macrocephalus Gunther 1864, Clarias batrachus (Linnaeus 1758), Paraplotosus albilabris (Valenciennes 1840), and Plotosus lineatus (Thunberg 1787), and the introduced Clarias gariepinus (Burchell 1822), Pangasianodon hypophthalmus (Sauvage 1878), Pterygoplichthys disjunctivus (Weber 1991), and Pterygoplichthys pardalis (Castelnau 1855). The DNA barcoding successfully discriminated seven of the eleven species; however, the COI was not able to differentiate Arius dispar from A. manillensis, and Pterygoplichthys disjunctivus from P. pardalis.

Several studies have been undertaken to determine the phylogenetic relationships of catfishes based on morphology (de Pinna 1998; Teugels 2003; Diogo 2004; Rodiles-Hernandez et al. 2005) as well as molecular data (Hardman 2005; Sullivan et al. 2006). Some issues and questions, however, remain on the classification of catfishes (Nelson 2006). For example, there is no consensus yet on the phylogenetic relationships of some catfish subfamilies, such as of that of Galeichthyinae and Ariinae (Kailola 2004; Betancur-R et al. 2007).

The mitochondrial gene regions COI, cytochrome b (cyt b), and 16S rRNA have been used to determine the phylogenetic relationships of catfishes. Wong et al. (2011) showed the phylogenetic relationships of nine catfish species of families Clariidae and Pangasiidae using COI. Guo et al. (2004) used both mitochondrial cyt b and 16S rRNA genes in elucidating the phylogeny and phylogeography of Chinese sisorid catfishes. Kartavtsev et al. (2007) also used cyt b and 16S rRNA regions in inferring the phylogeny of bullhead torrent catfish, Liobagrus obesus. The nuclear recombination activating gene (rag) is also used to infer deep phylogenetic relationships in fishes (Hardman 2004). Sullivan et al.

(2006) used both rag1 and rag2 in showing the monophyly of Siluriformes.

The combination of mitochondrial and nuclear genes tends to improve the accuracy of phylogenetic trees (Lake & Moore 1998). Information at different levels of phylogeny can be obtained because the mtDNA and nuclear genes have different evolutionary rates and modes of inheritance (Graybeal 1994). Mitochondrial genes can resolve terminal taxa because they evolve faster than nuclear genes (Avise 1994). Nuclear rag genes, on the other hand, are highly conserved (Hoofer et al. 2003); thus, they can show deep phylogenetic relationships (Sullivan et al. 2006).This study used the combination of the aforementioned three mtDNA and two nuclear genes in inferring the phylogeny of native and introduced catfishes in the Philippines.

The objective of this study was to determine the phylogenetic relationships of 11 species of catfishes belonging to Ariidae (Arius manillensis, A. dispar, and Plicofollis magatensis), Pangasiidae (Pangasianodon hypophthalmus), Clariidae (Clarias gariepinus, C. macrocephalus, and C. batrachus), Plotosidae (Paraplotosus albilabris and Plotosus lineatus), and Loricariidae (Pterygoplichthys disjunctivus and P. pardalis) using the mitochondrial genes COI, cyt b, 16S rRNA, and the nuclear genes rag1 and rag2.

MATERIALS AND METHODS

Sample collection and identification Eleven species of native and introduced catfishes were collected from around the Philippines (Table 1). These included the native Arius dispar, endemic Arius manillensis and Plicofollis magatensis (Ariidae), native Clarias batrachus and Clarias macrocephalus, introduced Clarias gariepinus (Clariidae), native Paraplotosus albilabris and Plotosus lineatus (Plotosidae), introduced Pangasianodon hypophthalmus (Pangasiidae), Pterygoplichthys disjunctivus and Pterygoplichthys pardalis (Loricariidae).The specimens were initially identified based on morphology. Arius dispar, Arius manillensis, and Plicofollis magatensis were identified following Herre (1926), Kailola (1999), and Marceniuk & Menezes (2007). Pangasianodon hypophthalmus was identified according to Roberts & Vidthayanon (1991). The Clarias species were identified using the taxonomic keys of Conlu (1986), Teugels et al. (1999), Sudarto & Pouyaud (2005), and Ng & Kottelat (2008). The two janitor fishes (Pterygoplichthys pardalis and P. disjunctivus) were identified according to Armbruster (2002) and Wu et al. (2011). Paraplotosus albilabris and

Philippine Journal of ScienceVol. 143 No. 2, December 2014

Yu and Quilang: Molecular Phylogeny of Philippines Catfishes

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Table 1. Taxonomy, collection site, G

enBank accession numbers and voucher ID

of specimens used in this study.

SpeciesFam

ilyStatus

Collection Site

No. of

specimens

GenB

ank accession number

Voucher ID

CO

Icyt b

16S rRN

Arag1

rag2

PlicofollisA

riidaeEndem

icC

amalaniugan,

2K

F604678;K

J533246;K

J533228;K

J533268;K

J533286;C

gA2;

magatensis

Cagayan

KF604677

KJ533247

KJ533229

KJ533269

KJ533287

CgA

3

Clarias

Clariidae

Native

Aparri,

2K

F604663;K

J533248;K

J533230;K

J533270;K

J533288;C

gCm

2;

macrocephalus

Cagayan

KF604666

KJ533249

KJ533231

KJ533271

KJ533289

CgC

m3

Clarias

Clariidae

Native

Aparri,

2K

F604645;K

J533250;K

J533232;K

J533272;K

J533290;C

CB

R1;

batrachusC

agayanK

F604646K

J533251K

J533233K

J533273K

J533291C

CB

R2

Clarias

Clariidae

IntroducedB

ustos,2

KF604660;

KJ533252;

KJ533234;

KJ533274;

KJ533292;

BC

G1;

gariepinusB

ulacanK

F604661K

J533253K

J533235K

J533275K

J533293B

CG

2

PangasianodonPangasiidae

IntroducedC

abaritan, Bay

2K

F604668;K

J533254;K

J533236;K

J533276;K

J533294;LPh1;

hypophthalmus

town, Laguna

KF604667

KJ533255

KJ533237

KJ533277

KJ533295

LPh2

ParaplotosusPlotosidae

Native

Dum

angas,2

KF604673;

KJ533256;

KJ533238;

KJ533278;

KJ533296;

SPa1;

albilabrisIloilo

KF604672

KJ533257

KJ533239

KJ533279

KJ533297

SPa2

PlotosusPlotosidae

Native

Pagbilao, Quezon

2K

F604684;K

J533258;K

J533240;K

J533280;K

J533298;Q

P1;

lineatusProvince

KF604690

KJ533259

KJ533241

KJ533281

KJ533299

QP2

PterygoplichthysLoricariidae

IntroducedTanay, R

izal,2

KF604691;

KJ533260;

KJ533242;

KJ533282;

KJ533300;

TRPd1;

disjunctivusLaguna de B

ayK

F604692K

J533261K

J533243K

J533283K

J533301TR

Pd2

PterygoplichthysLoricariidae

IntroducedTanay, R

izal2

KF604698;

KJ533262;

KJ533244;

KJ533284;

KJ533302;

TRPp1;

pardalisLaguna de B

ayK

F604699K

J533263K

J533245K

J533285K

J533303TR

Pp2

Arius A

riidaeEndem

icTanza, C

avite,2

KF604641;

KJ533154;

KJ533174;

KJ533264;

KJ533214;

TCA

m1;

manillensis

Manila B

ayK

F604642K

J533155K

J533175K

J533265K

J533215TC

Am

2

Arius A

riidaeN

ativeTanay R

izal,2

KJ533143;

KJ533165;

KJ533185;

KJ533266;

KJ533225;

TRA

d1;

dispar Laguna de B

ayK

J533144K

J533166K

J533186K

J533267K

J533226TR

Ad2



189

Plotosus lineatus were identified following Herre (1926), Allen (1998), and Ferraris (1999). Molecular identification of the specimens was also done using DNA barcoding (Quilang & Yu 2013). All specimens were preserved in 10% formalin and deposited in the Institute of Biology, University of the Philippines, Diliman.

DNA extraction and PCR AmplificationDNA was extracted from a white muscle tissue sample (approximately 20 mg) using the Promega Wizard® Genomic DNA purification kit (Madison, WI, USA) following manufacturer’s protocol. A total of 22 specimens, two specimens per species, were used. COI, cyt b,16S rRNA, rag1, and rag2 genes were amplified using the primers listed in Table 2.

The primers FishF1, FishF2, FishR1 and FishR2 (Ward et al. 2005), were used to amplify 615 bp of COI. The newly designed primers LSILCB0I and SILCBO3, and primers developed by Wu et al. (2011), L1 and H2, were used to amplify approximately 1112 bp of cyt b. The thermocycler conditions for the amplification of COI and cyt b were as follows (Ward et al. 2005): initiation for 2 min at 95oC, 35 cycles of denaturation for 0.5 min at 94oC, primer annealing for 0.5 min at 54oC, primer extension for 1 min at 72oC, and final extension for 10 min at 72oC.

The primers developed by Palumbi (1996), 16Sar and 16Sbr, were used for the amplification of approximately

565 bp of 16S rRNA. The thermocycler conditions were as follows: initiation for 3.0 min at 94oC, 43 cycles of denaturation for 0.5 min at 94oC, primer annealing for 0.5 min at 45oC, primer extension for 1 min at 72oC, and final extension for 5 min at 72oC.

The F1483I and R3055 primers (López et al. 2004), and F1514 and R3026 primers (Sullivan et al. 2006) were used to amplify rag1 (approximately 1425bp). The MHF1 and MHR1 primers designed by Hardman (2004), and the FARIRag2 and RARIRag2 primers designed in this study, were used for rag2, which amplified approximately 955 bp. The thermocycler conditions in Sullivan et al. (2006) were modified and used to amplify both rag genes. These conditions were as follows: initiation for 1.0 min at 94oC, 35 cycles of denaturation for 0.5 min at 94oC, primer annealing for 0.5 min at 52-57oC, primer extension for 2 min at 72oC, and final extension for 10 min at 72oC.

All PCR master mixes had the same ingredients for a 50-µL polymerase chain reaction (PCR): the reaction consisted of 1.0 µL of 0.05 mM dNTP, 2.5 µL of 0.1mM of each primer, 5.0 µL of 1x PCR buffer, 0.5 µL of (1.25U) Taq polymerase (Roche TaqdNTPack), 34.5 µL of ultrapure water and 4.0 µL of DNA template.

PCR products were viewed on one percent agarose gel with ethidium bromide. Bands from the gel correctly matching the expected size of the fragment were excised and then purified using Qiaquick® Gel Extraction Kit (Qiagen,

Table 2. List of Primers used in this study.

Primer Gene Sequence (5’ to 3’) Source

FishF1 COI TCAACCAACCACAAAGACATTGGCAC Ward el al. (2005)

FishF2 COI TCGACTAATCATAAAGATATCGGCAC Ward el al. (2005)

FishR1 COI TAGACTTCTGGGTGGCCAAAGAATCA Ward el al. (2005)

FishR2 COI ACTTCAGGGTGACCGAAGAATCAGAA Ward el al. (2005)

LSILCB0I cyt b TAACCAGGACTAATGACTTG This study

SILCBO3 cyt b AAGACCGGCGCTTTAAGCTA This study

L1 cyt b AAATACGGCGCAGGATTAGAAGCAAC Wu et al. (2011)

H2 cyt b GGGAGTTAAAATCTCTCTTTTCTGGC Wu et al. (2011)

16Sar 16S rRNA CGCCTGTTTATCAAAAACAT Palumbi (1996)

16Sbr 16S rRNA CGGTCTGAACTCAGATCACGT Palumbi (1996)

F1483I rag1 CTCAGCTGTAGCCAGTACCACAAAATG López et al. (2004)

R3055 rag1 TGAGCCTCCATGAACTTCTGAAGrTAyTT López et al. (2004)

F1514 rag1 CGCACkGTTAAAGCTATkAGTGGGCG Sullivan et al. (2006)

R3026 rag1 GATGTGTACAGCCAGTGGTGTTTTAAT Sullivan et al. (2006)

MHF1 rag2 TGyTATCTCCCACCTCTGCGyTACC Hardman (2004)

MHR1 rag2 TCATCCTCCTCATCkTCCTCwTTGTA Hardman (2004)

FARIRag2 rag2 CCAACAACGAGCTGTCCTCA This study

RARIRag2 rag2 GCTGAATCCTCAAAATCAGTGG This study



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Valencia, CA, USA) following the manufacturer’s instructions. The purified DNA products were sent to 1st BASE in Selangor Darul Ehsan, Malaysia for bidirectional sequencing.

DNA sequence analysisIn each specimen, the consensus sequence of each gene was obtained by aligning the sequences generated using forward and reverse primers using the Staden Package v4.10 (Staden et al. 2000).There is no species of Siluriformes in GenBank with sequences for all the five genes used in this study; thus, three species of the order Characiformes, Lophiobrycon weitzmani, Kolpotocheirodon theloura, and Serrapinnus calliurus, were used as outgroups (Table 3). The choice of an outgroup is based on past studies showing that Siluriformes, Characiformes, and Gymnotiformes are closely related and formed the Characiphysi clade (Dimmick & Larson 1996; Saitoh et al. 2003). The sequences were aligned and analyzed using BioEdit sequence alignment version 7.0.5.3 (Hall 1999). Unique sequences were determined using Data Analysis in Molecular Evolution (DAMBE) version 5.2.57 (Xia 2013) and used for the subsequent analyses. PartitionFinder (Lanfear et al. 2012) was used to detect the appropriate model for each partition based on corrected Akaike Information Criterion (AICc) (Posada

2008). The partitions were set with respect to gene and codon positions. The appropriate model for each partition is shown in Table 4.

Xia test (Xia & Lemey 2009; Xia 2013) was implemented in DAMBE to check for substitution oversaturation based on the concept of entropy information theory (Xia et al. 2003). Phylogenetic analyses were performed using the model based Maximum Likelihood (ML) and Bayesian inference (BI). GARLI 2.0 (Zwickl 2006; Bazinet et al. 2014) and MrBayes 3.2 (Ronquist & Huelsenbeck 2003) were used for the construction of ML and BI tree, respectively. GARLI 2.0 (Zwickl 2006; Bazinet et al. 2014) was performed under the best-fit model estimated with the AICc in PartitionFinder (Lanfear et al. 2012), and the nonparametric bootstrap analysis was determined with 1000 replicates. The number of generations to terminate searches when no topological improvement were found was adjusted to 75,000 generations (genthreshfortopoterm=75,000). For the Bayesian inference, the number of substitution parameters (1, 2 or 6), gamma-shape parameter (equal or gamma), and number of gamma category (16 if the distributed rates is gamma) were set according to the selected model using AICc in PartitionFinder. Markov Chain Monte Carlo (MCMC) analyses were conducted with 10,000,000 generations using the optimized temperature resulting in

Table 3. List of sequences downloaded from GenBank and used as outgroup in this study.Species Voucher COI 16S rRNA cyt b rag1 rag2 Source

Lophiobrycon weitzmani LBP 1225 GU701436 HQ171411 HQ289698 HQ289312 HQ289504

Oliveira et al. (2011);Pereira et al. (2013)

Kolpotocheirodontheloura LBP 5033 HM376391 HQ171336 HQ289625 HQ289238 HQ289432


Serrapinnus calliurus LBP 3731 HM371155 HQ171291 HQ289580 HQ289195 HQ289388


Table 4. Substitution models for nucleotide data partitions selected using the AICc in PartitionFinder (Lanfear et al. 2012).

PartitionFinder ModelCOI 1st codon GTR+I

COI 2nd codon F81

COI 3rd codon, cyt b 3rd codon, rag1 1st codon, rag2 2nd codon GTR+G

cyt b 1st codon, rag1 3rd codon, rag2 1st codon SYM+G

cyt b 2nd codon, rag1 2nd codon, HKY+I

16S rRNA GTR+I+G

rag2 3rd codon K80+I



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an acceptance rate of the Metropolis-Hastings MCMC sampler ranging between 0.1 and 0.7. The marginal probability, consensus phylograms, and posterior probabilities of nodes were estimated from the post-burn-in samples, with 19001 burn-in value for 10,000,000 generations.

All DNA sequences generated from this study have been submitted to GenBank (Table 1).

RESULTSA total of 110 sequences were generated from five gene regions of the 11 native and introduced catfishes in the Philippines. A 615-bp COI sequence, 958-bp cyt b, 581-bp 16S rRNA, 1238-bp rag1, and 824-bp rag2 sequence were concatenated, totaling to 4216 bp. Data Analysis in Molecular Evolution (DAMBE) version 5.2.57 (Xia 2013) yielded 21 unique sequences from 25 concatenated sequences.

The concatenated sequences yielded 1526 (36%) variable characters and 1385 (33%) parsimony-informative characters out of 4216 characters. Xia test to determine substitution saturation yielded an lss<lss.c, in which lss values are significantly lower (P<0.05) than lss.c in all gene regions based on three codon positions. These indicate that there was no substantial substitution saturation. The appropriate model for each gene and codon position in PartitonFinder (Lanfear 2012) was summarized in Table 4. These models were implemented for the ML and BI analyses.

The two trees generated (ML, BI) showed that all 11 species grouped according to their genera and families (Figure 1). Arius manillensis, A. dispar, and Plicofollis magatensis (Ariidae) clustered together with 100% bootstrap support in ML tree and 1.0 Bayesian posterior probability in BI tree. Pangasianodon hypophthalmus (Pangasiidae) clustered with 100% bootstrap support in ML tree and 1.0 Bayesian posterior probability in BI tree. Clarias gariepinus, C. macrocephalus, and C. batrachus (Clariidae) formed a clade with 100% bootstrap supporting ML tree and 1.0 Bayesian posterior probability in BI tree. Clarias macrocephalus and C. batrachus were more closely related to each other, than either is to C. gariepinus. Two species of the family Loricariidae (Pterygoplichthys disjunctivus and P. pardalis also formed a single cluster with 100% bootstrap supporting ML tree and 1.0 Bayesian posterior probability in BI tree. Paraplotosus albilabris and Plotosus lineatus (Plotosidae) also grouped together with 100% bootstrap support in ML tree and 1.0 Bayesian posterior probability in BI tree. The three sequences of Characiformes (outgroup) also formed a cluster with 100%

bootstrap support and 1.0 Bayesian posterior probability.

The topology of the trees was the same for ML and BI (Figure 1). Pangasianodon hypophthalmus of Pangasiidae, and Plotosidae formed a clade with 90% bootstrap support in the ML and 1.0 Bayesian posterior probability in BI tree. This latter clade is the sister group of Ariidae with 100%bootstrap support and 1.0 Bayesian posterior probability in ML and BI tree, respectively (Figure 1). The clade of the three families (Pangasiidae, Ariidae and Plotosidae) is the sister group of Clariidae. The clade formed by these four families, Pangasiidae, Ariidae, Plotosidae and Clariidae, is the sister group of Loricariidae.

DISCUSSION

Monophyly of each familyAll 11 species grouped according to their genera and families. Clarias gariepinus, C. macrocephalus, and C. batrachus (Clariidae) formed a monophyletic clade. Clariidae (walking catfishes) is a unique clade by having an arborescent suprabranchial organ (Agnese & Teugels 2005). The monophyly of this family is supported by Agnese & Teugels (2005), Sullivan et al. (2006) and Pouyaud et al. (2009). Agnese & Teugels (2005) used cyt b while Sullivan et al. (2006) used rag1 and rag2, and Pouyaud et al. (2009) used cyt b, 16S rRNA and 29 morphometric measurements.

The genus Pangasianodon (Pangasiidae), commonly known as shark catfishes (Pouyaud et al. 2000) belongs to a monophyletic family (Sullivan et al. 2006) and is characterized by maxillary and mandibulary pairs of barbels, well developed adipose and anal fin, one or two spines on a short dorsal fin, and laterally compressed body (Teugels 1996).

Plotosus lineatus and Paraplotosus albilabris formed a monophyletic cluster. Plotosidae (eel catfishes) is monophyletic and possesses six autapomorphies based on osteology and myology (Oliveira et al. 2001; Sullivan et al. 2006). This family is commonly considered a monophyletic group by several authors (Mo 1991; Oliveira et al. 2001; Sullivan et al. 2006).

Arius manillensis, A. dispar, and Plicofollis magatensis (Ariidae) (sea catfishes) formed a monophyletic clade. The monophyly of Ariidae is strongly supported by previous phylogenetic studies using morphological and molecular data (Mo 1991; Kailola 2004; Hardman2005; Sullivan et al. 2006; Betancur-R et al. 2007; Marceniuk et al. 2012). The monophyly of Ariidae was first claimed by Mo (1991) based on osteological evidences. Oliveira et al. (2002) and



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Figure 1. Rooted Maximum Likelihood (ML) and Bayesian Inference (BI) consensus tree of 25 concatenated cytochrome c oxidase I (COI), cytochrome b (cyt b), 16S rRNA, recombination activating genes 1 and 2 (rag1 and rag2) sequences from 22 Philippine catfishes (Siluriformes) and three Characiformes (outgroup taxon). Bootstrap support of 1,000 replicates and Bayesian posterior probabilities are shown (ML/BI) at the branches. Bar indicates substitutions per site. Specimen vouchers are indicated for each species (letters and numbers after each bar). Status, “N” for native, and “I” for introduced species in the Philippines are indicated in parenthesis. Analyses were conducted using GARLI 2.0 (Zwickl 2006; Bazinet et al. 2014) for ML and MrBayes 3.2 (Ronquist and Huelsenbeck 2003) for BI under the best-fit model estimated with the AICc in PartitionFinder (Lanfear et al. 2012).

Kailola (2004) showed synapomorphies of the family. In 2005, Diogo was able to find evidence of monophyly of this family based on osteological and myological characters. Hardman (2005), Betancur-R et al. (2007) and Sullivan et al. (2006) also support the monophyly of this family based on molecular data.

The two species of Loricariidae (suckermouth armored catfishes), Pterygoplichthys disjunctivus and P. pardalis, formed a monophyletic cluster. Loricariidae is characterized

by having an armored body and a sucker-like ventrally situated mouth (Howes 1983).The monophyly of this family was also shown by Cramer et al. (2011) based on COI, rag1 and rag2. In addition, there are autapomorphies to support the monophyly of this family: these include a lack of hyomandibulae fossae, nasal capsule confined to lateral ethmoid, body encased in scutes, and the dilatator opercula muscle is antero-ventrally oriented with presence of retractor palatine (Howes 1983).



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Possible synonymy of two Arius species and of two Pterygoplichthys species The concatenation of five gene regions was not able to discriminate between Arius manillensis and A. dispar, and between Pterygoplichthys disjunctivus and P. pardalis (Figure 1). This supports previous DNA barcoding studies, which suggest the possibility that the two species of Arius and the two species of Pterygoplichthys are just synonymous (Jumawan et al. 2011; Santos & Quilang 2011; Quilang & Yu 2013). The mean COI genetic divergences between the two Arius species were low in the study of Santos & Quilang (2011) and Quilang & Yu (2013). Low genetic divergence between the two Pterygoplichthys species was also reported by Wu et al. (2011) and Quilang & Yu (2013) based on cyt b and COI, respectively. The only morphological trait that can be used to distinguish between the two Arius species is the pattern of tooth patch on the palate: A. manillensis has two large ovate tooth patches both on the upper and lower parts of the palate, while A. dispar has two widely separated tooth patches on the upper palate only (Kailola 1999). On the other hand, vermiculations on the ventral side are used to distinguish between the two Pterygoplichthys species: P. disjunctivus has continuous and curved dark lines on a white background, whereas P. pardalis has dark spots on a white background (Chavez et al. 2006). These traits may not be good characters to differentiate the two Arius species and the two Pterygoplichthys species (Wu et al. 2011; Quilang & Yu 2013).

Phylogenetic relationships of the five familiesThe ML and BI trees generated a (Loricariidae + (Clariidae + (Ariidae + (Pangasiidae + Plotosidae)))) topology (Figure 1). This topology is different from the past topology generated by Mo (1991), de Pinna (1998) and Diogo (2004) based on morphology. Mo’s (1991) topologies differed on Ariidae, Pangasiidae and Plotosidae relationship, while de Pinna (1998) and Diogo (2004) differed on Clariidae, Ariidae, Pangasiidae and Plotosidae relationships. The conflict between the morphological and molecular topology can be attributed to homoplasy (Davalos et al. 2012). Mo (1991) used 126 morphological characters in his analysis and had a Consistency Index (CI) of 0.36. de Pinna’s (1998) work is based on 239 morphological characters and had a CI of 0.41, while Diogo (2004) used 440 morphological characters and had CI of 0.52. On the other hand, the concatenated data in this study had a CI of 0.64. Consistency Index (CI) is inversely proportion to homoplasy index (Diogo 2007).These indicate that the phylogenetic topologies based on morphology had higher homoplasy than the phylogenetic topology generated in this study. Also, catfish is a very diverse group that is highly homoplasic (Diogo 2007). Past morphological studies might neglect to include this

homoplasy resulting in incongruence between molecular and morphological trees (Diogo 2004).

Loricariidae is separated from the monophyletic clade of Ariidae, Pangasiidae, Clariidae and PlotosidaeCatfishes are divided into two distinct suborders, the Loricarioidei and the Siluroidei. The family Loricariidae belongs to the suborder Loricarioidei, while four other catfish families in this study, namely, Ariidae, Pangasiidae, Clariidae and Plotosidae belong to the suborder Siluroidei (Armbruster 2011). Each of the two suborders formed a monophyletic clade (Sullivan et al. 2006; Armbruster 2011). The consensus of the studies based on morphology (de Pinna 1998; Diogo 2004) and molecular data (Hardman 2005; Sullivan et al. 2006) is that Loricariidae is separated from the clade consisting of Ariidae, Pangasiidae, Clariidae and Plotosidae. The topology of the trees generated in this study (Figure 1) is consistent with this consensus.

The Loricarioidei was grouped by the derived presence of odontodes or integumentary teeth (Armbruster 2011) and no doubt a monophyletic suborder (Bailey& Baskin 1976; Mo 1991; de Pinna 1998; Hardman 2005; Sullivan et al. 2006). Howes (1983) diagnosed the six families (Loricariidae, Astroblepidae, Scoloplacidae, Callichthyidae, Trichomycteridae, Nematogenyidae) under the Loricarioidei by the presence of encapsulated swimbladder that is divided into separate vesicles, in which some part of the cranium contributes to encapsulation.

Clariidae is separated from the monophyletic clade of Ariidae, Pangasiidae and PlotosidaeAnalyses show that Clariidae is separated from the monophyletic clade of Ariidae, Pangasiidae and Plotosidae (Figure 1). Mo (1991) produced two cladograms based on numerical analysis of 126 morphological characters. The first cladogram unweighted the 126 morphological characters, while the second cladogram weighted the 126 morphological characters. Both the cladograms of Mo (1991) and Hardman (2005) showed that Loricariidae is more closely related to Clariidae than to the other three families (Ariidae, Pangasiidae and Plotosidae).

CONCLUSION AND RECOMMENDATIONSAnalyses (ML and BI) support the monophyly of each of the five catfish families. All topologies showed that Loricariidae is separated from the monophyletic clade formed by the other four catfish families (Ariidae, Clariidae, Pangasiidae, and Plotosidae). Clariidae



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is separated from the monophyletic clade formed by the three catfish families Ariidae, Pangasiidae, and Plotosidae ML and BI analyses gave (Ariidae + (Pangasiidae + Plotosidae)) topology. Also, concatenated sequences of specimens of each of the two species of Arius could not be discriminated from each other. This is also the case for the two species of Pterygoplichthys. It is possible that the two species of Arius and the two species of Pterygoplichthys are synonymous. Future studies may use cytogenomic markers such as microsatellites (Pereira et al. 2014) to establish if the two Arius species and the two Pterygoplichthys species are the same or different species. Future studies may also combine molecular and morphological methods in inferring phylogenetic relationships. The combination of two may improve the partition branch support values (Baker et al. 1998) and remove homoplasy in individual data partitions (Farris 1983).

ACKNOWLEDGMENTSWe would like to thank the Office of the Chancellor of the University of the Philippines Diliman, in collaboration with the Office of the Vice Chancellor for Research and Development, for funding support through the PhD Incentive Awards (Project No. 111105 PhDIA) given to J. P. Quilang. Thanks also to the Department of Science and Technology – Science Education Institute (DOST-SEI) Accelerated Science and Technology Human Resource Development Program (ASTHRDP) for scholarship and additional funding support given to Shiny Cathlynne S. Yu.

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