Philippine Journal of Science 143 (2): 187-198, December 2014 ISSN 0031 - 7683 Date Received: 02 February 2014

Molecular Phylogeny of (Teleostei: Siluriformes) in the Philippines Using the Mitochondrial Genes COI, Cyt b, 16S rRNA, and the Nuclear Genes Rag1 and Rag2

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

INTRODUCTION Pterygoplichthys disjunctivus and Pterygoplichthys pardalis are causing environmental problems. Catfishes (Order Siluriformes) are a diverse group of ray-finned fishes (Nelson 2006) that are distributed in Catfishes are primarily freshwater fishes with only all continents (Diogo 2004) and comprise more than two marine families: Plotosidae, which is distributed 3,088 valid species distributed among 477 genera and in the Indo-West Pacific and Ariidae, which is found 36 families (Ferraris 2007). Catfishes are valued as worldwide in tropical to warm temperate zones (Kailola popular sport fish, food items, and tropical aquarium fish 2004). In the Philippines, Froese & Pauly (2013) in (Nelson 2006). Two introduced species, namely, Clarias FishBase list 31 species of catfishes belonging to eight gariepinus and Pangasianodon hypophthalmus are used in families, namely, Ariidae, Clariidae, Callichthyidae, aquaculture, while two other introduced species, namely, Loricariidae, Ictaluridae, Pangasiidae, Plotosidae, and Siluridae. However, Corydoras aeneus, the single species *Corresponding author: [email protected] of Callichthyidae, is questionable as it was only reported

187 Philippine Journal of Science Yu and Quilang: Molecular Phylogeny of Vol. 143 No. 2, December 2014 Philippines Catfishes as a specimen in a fish living museum (Froese & Pauly (2006) used both rag1 and rag2 in showing the monophyly 2013). Likewise, the occurrence of Ictalurus punctatus, of Siluriformes. the single species of Ictaluridae, is questionable as this species was introduced in 1974 but it could not thrive in The combination of mitochondrial and nuclear genes the natural conditions of the Philippines (Juliano et al. tends to improve the accuracy of phylogenetic trees 1989). Only 15 species do not have contradicting (Lake & Moore 1998). Information at different levels reports and are certain to occur in the Philippines. These of phylogeny can be obtained because the mtDNA and include Arius dispar, Arius manillensis, Plicofollis nuclear genes have different evolutionary rates and modes magatensis, Plicofollis nella, Clarias batrachus, Clarias of inheritance (Graybeal 1994). Mitochondrial genes can gariepinus, Clarias macrocephalus, Clarias nieuhofii, resolve terminal taxa because they evolve faster than Pterygoplichthys disjunctivus, Pterygoplichthys pardalis, nuclear genes (Avise 1994). Nuclear rag genes, on the Pangasianodon hypophthalmus, Paraplotosus albilabris, other hand, are highly conserved (Hoofer et al. 2003); Plotosus canius, Plotosus lineatus, and Pterocryptis thus, they can show deep phylogenetic relationships taytayensis. Eleven species of the catfishes reported (Sullivan et al. 2006).This study used the combination of in the Philippines have been DNA barcoded using the the aforementioned three mtDNA and two nuclear genes in cytochrome c oxidase subunit I (COI) gene (Quilang inferring the phylogeny of native and introduced catfishes & Yu 2013). These species include the endemic in the Philippines. Arius manillensis Valenciennes 1840 and Plicofollis The objective of this study was to determine the magatensis (Herre 1926), the native Arius dispar Herre phylogenetic relationships of 11 species of catfishes 1926, Clarias macrocephalus Gunther 1864, Clarias belonging to Ariidae (Arius manillensis, A. dispar, and batrachus (Linnaeus 1758), Paraplotosus albilabris Plicofollis magatensis), Pangasiidae (Pangasianodon (Valenciennes 1840), and Plotosus lineatus (Thunberg hypophthalmus), Clariidae (Clarias gariepinus, 1787), and the introduced Clarias gariepinus (Burchell C. macrocephalus, and C. batrachus), Plotosidae 1822), Pangasianodon hypophthalmus (Sauvage 1878), (Paraplotosus albilabris and Plotosus lineatus), and Pterygoplichthys disjunctivus (Weber 1991), and Loricariidae (Pterygoplichthys disjunctivus and P. Pterygoplichthys pardalis (Castelnau 1855). The DNA pardalis) using the mitochondrial genes COI, cyt b, 16S barcoding successfully discriminated seven of the eleven rRNA, and the nuclear genes rag1 and rag2. 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 MATERIALS AND METHODS the phylogenetic relationships of catfishes based on morphology (de Pinna 1998; Teugels 2003; Diogo 2004; Sample collection and identification Rodiles-Hernandez et al. 2005) as well as molecular Eleven species of native and introduced catfishes data (Hardman 2005; Sullivan et al. 2006). Some issues were collected from around the Philippines (Table and questions, however, remain on the classification 1). These included the native Arius dispar, endemic of catfishes (Nelson 2006). For example, there is no Arius manillensis and Plicofollis magatensis (Ariidae), consensus yet on the phylogenetic relationships of some native Clarias batrachus and Clarias macrocephalus, catfish subfamilies, such as of that of Galeichthyinae and introduced Clarias gariepinus (Clariidae), native Ariinae (Kailola 2004; Betancur-R et al. 2007). Paraplotosus albilabris and Plotosus lineatus (Plotosidae), introduced Pangasianodon hypophthalmus (Pangasiidae), The mitochondrial gene regions COI, cytochrome b Pterygoplichthys disjunctivus and Pterygoplichthys (cyt b), and 16S rRNA have been used to determine pardalis (Loricariidae).The specimens were initially the phylogenetic relationships of catfishes. Wong et al. identified based on morphology. Arius dispar, Arius (2011) showed the phylogenetic relationships of nine manillensis, and Plicofollis magatensis were identified catfish species of families Clariidae and Pangasiidae following Herre (1926), Kailola (1999), and Marceniuk using COI. Guo et al. (2004) used both mitochondrial cyt & Menezes (2007). Pangasianodon hypophthalmus b and 16S rRNA genes in elucidating the phylogeny and was identified according to Roberts & Vidthayanon phylogeography of Chinese sisorid catfishes. Kartavtsev (1991). The Clarias species were identified using the et al. (2007) also used cyt b and 16S rRNA regions taxonomic keys of Conlu (1986), Teugels et al. (1999), in inferring the phylogeny of bullhead torrent catfish, Sudarto & Pouyaud (2005), and Ng & Kottelat (2008). Liobagrus obesus. The nuclear recombination activating The two janitor fishes (Pterygoplichthys pardalis and P. gene (rag) is also used to infer deep phylogenetic disjunctivus) were identified according to Armbruster relationships in fishes (Hardman 2004). Sullivan et al. (2002) and Wu et al. (2011). Paraplotosus albilabris and

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Table 1. , collection site, GenBank accession numbers and voucher ID of specimens used in this study.

No. of Species Family Status Collection Site GenBank accession number Voucher ID

Philippines Catfishes Philippines specimens

COI cyt b 16S rRNA rag1 rag2 Plicofollis Ariidae Endemic Camalaniugan, 2 KF604678; KJ533246; KJ533228; KJ533268; KJ533286; CgA2; magatensis Cagayan KF604677 KJ533247 KJ533229 KJ533269 KJ533287 CgA3 Clarias Aparri, KF604663; KJ533248; KJ533230; KJ533270; KJ533288; CgCm2; Yu and Quilang: Molecular Phylogeny of Phylogeny Molecular and Quilang: Yu Clariidae Native 2 macrocephalus Cagayan KF604666 KJ533249 KJ533231 KJ533271 KJ533289 CgCm3 Clarias Aparri, KF604645; KJ533250; KJ533232; KJ533272; KJ533290; CCBR1; Clariidae Native 2 batrachus Cagayan KF604646 KJ533251 KJ533233 KJ533273 KJ533291 CCBR2 Clarias Bustos, KF604660; KJ533252; KJ533234; KJ533274; KJ533292; BCG1; Clariidae Introduced 2 gariepinus Bulacan KF604661 KJ533253 KJ533235 KJ533275 KJ533293 BCG2 Pangasianodon Cabaritan, Bay KF604668; KJ533254; KJ533236; KJ533276; KJ533294; LPh1; Pangasiidae Introduced 2 hypophthalmus town, Laguna KF604667 KJ533255 KJ533237 KJ533277 KJ533295 LPh2 Paraplotosus Dumangas, KF604673; KJ533256; KJ533238; KJ533278; KJ533296; SPa1; Plotosidae Native 2 albilabris Iloilo KF604672 KJ533257 KJ533239 KJ533279 KJ533297 SPa2 Plotosus Pagbilao, Quezon KF604684; KJ533258; KJ533240; KJ533280; KJ533298; QP1; Plotosidae Native 2 lineatus Province KF604690 KJ533259 KJ533241 KJ533281 KJ533299 QP2 Pterygoplichthys Tanay, Rizal, KF604691; KJ533260; KJ533242; KJ533282; KJ533300; TRPd1; Loricariidae Introduced 2 disjunctivus Laguna de Bay KF604692 KJ533261 KJ533243 KJ533283 KJ533301 TRPd2 Pterygoplichthys Tanay, Rizal KF604698; KJ533262; KJ533244; KJ533284; KJ533302; TRPp1; Loricariidae Introduced 2 pardalis Laguna de Bay KF604699 KJ533263 KJ533245 KJ533285 KJ533303 TRPp2 Arius Tanza, Cavite, KF604641; KJ533154; KJ533174; KJ533264; KJ533214; TCAm1; Ariidae Endemic 2 manillensis Manila Bay KF604642 KJ533155 KJ533175 KJ533265 KJ533215 TCAm2 Arius Tanay Rizal, KJ533143; KJ533165; KJ533185; KJ533266; KJ533225; TRAd1; Ariidae Native 2 dispar Laguna de Bay KJ533144 KJ533166 KJ533186 KJ533267 KJ533226 TRAd2 Philippine Journal of Science Journal Philippine December 2014 143 No. 2, Vol. Philippine Journal of Science Yu and Quilang: Molecular Phylogeny of Vol. 143 No. 2, December 2014 Philippines Catfishes

Plotosus lineatus were identified following Herre (1926), 565 bp of 16S rRNA. The thermocycler conditions were Allen (1998), and Ferraris (1999). Molecular identification as follows: initiation for 3.0 min at 94oC, 43 cycles of of the specimens was also done using DNA barcoding denaturation for 0.5 min at 94oC, primer annealing for (Quilang & Yu 2013). All specimens were preserved in 0.5 min at 45oC, primer extension for 1 min at 72oC, and 10% formalin and deposited in the Institute of Biology, final extension for 5 min at 72oC. University of the Philippines, Diliman. The F1483I and R3055 primers (López et al. 2004), and F1514 and R3026 primers (Sullivan et al. 2006) were used DNA extraction and PCR Amplification to amplify rag1 (approximately 1425bp). The MHF1 and DNA was extracted from a white muscle tissue sample MHR1 primers designed by Hardman (2004), and the (approximately 20 mg) using the Promega Wizard® FARIRag2 and RARIRag2 primers designed in this study, Genomic DNA purification kit (Madison, WI, USA) were used for rag2, which amplified approximately 955 following manufacturer’s protocol. A total of 22 bp. The thermocycler conditions in Sullivan et al. (2006) specimens, two specimens per species, were used. COI, were modified and used to amplify both rag genes. These cyt b,16S rRNA, rag1, and rag2 genes were amplified conditions were as follows: initiation for 1.0 min at 94oC, using the primers listed in Table 2. 35 cycles of denaturation for 0.5 min at 94oC, primer annealing for 0.5 min at 52-57oC, primer extension for The primers FishF1, FishF2, FishR1 and FishR2 (Ward et 2 min at 72oC, and final extension for 10 min at 72oC. al. 2005), were used to amplify 615 bp of COI. The newly designed primers LSILCB0I and SILCBO3, and primers All PCR master mixes had the same ingredients for a developed by Wu et al. (2011), L1 and H2, were used to 50-µL polymerase chain reaction (PCR): the reaction amplify approximately 1112 bp of cyt b. The thermocycler consisted of 1.0 µL of 0.05 mM dNTP, 2.5 µL of 0.1mM conditions for the amplification of COI and cyt b were as of each primer, 5.0 µL of 1x PCR buffer, 0.5 µL of follows (Ward et al. 2005): initiation for 2 min at 95oC, (1.25U) Taq polymerase (Roche TaqdNTPack), 34.5 µL 35 cycles of denaturation for 0.5 min at 94oC, primer of ultrapure water and 4.0 µL of DNA template. annealing for 0.5 min at 54oC, primer extension for 1 min at 72oC, and final extension for 10 min at 72oC. PCR products were viewed on one percent agarose gel with ethidium bromide. Bands from the gel correctly matching The primers developed by Palumbi (1996), 16Sar and the expected size of the fragment were excised and then 16Sbr, were used for the amplification of approximately 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 2008). The partitions were set with respect to gene and instructions. The purified DNA products were sent to 1st codon positions. The appropriate model for each partition BASE in Selangor Darul Ehsan, Malaysia for bidirectional is shown in Table 4. sequencing. Xia test (Xia & Lemey 2009; Xia 2013) was implemented in DAMBE to check for substitution oversaturation based DNA sequence analysis on the concept of entropy information theory (Xia et al. In each specimen, the consensus sequence of each 2003). Phylogenetic analyses were performed using the gene was obtained by aligning the sequences generated model based Maximum Likelihood (ML) and Bayesian using forward and reverse primers using the Staden inference (BI). GARLI 2.0 (Zwickl 2006; Bazinet et Package v4.10 (Staden et al. 2000).There is no species al. 2014) and MrBayes 3.2 (Ronquist & Huelsenbeck of Siluriformes in GenBank with sequences for all 2003) were used for the construction of ML and BI the five genes used in this study; thus, three species tree, respectively. GARLI 2.0 (Zwickl 2006; Bazinet of the order Characiformes, Lophiobrycon weitzmani, et al. 2014) was performed under the best-fit model Kolpotocheirodon theloura, and Serrapinnus calliurus, estimated with the AICc in PartitionFinder (Lanfear were used as outgroups (Table 3). The choice of an et al. 2012), and the nonparametric bootstrap analysis outgroup is based on past studies showing that Siluriformes, was determined with 1000 replicates. The number of Characiformes, and Gymnotiformes are closely related generations to terminate searches when no topological and formed the Characiphysi clade (Dimmick & Larson improvement were found was adjusted to 75,000 1996; Saitoh et al. 2003). The sequences were aligned generations (genthreshfortopoterm=75,000). For the and analyzed using BioEdit sequence alignment version Bayesian inference, the number of substitution parameters 7.0.5.3 (Hall 1999). Unique sequences were determined (1, 2 or 6), gamma-shape parameter (equal or gamma), using Data Analysis in Molecular Evolution (DAMBE) and number of gamma category (16 if the distributed version 5.2.57 (Xia 2013) and used for the subsequent rates is gamma) were set according to the selected model analyses. PartitionFinder (Lanfear et al. 2012) was used using AICc in PartitionFinder. Markov Chain Monte to detect the appropriate model for each partition based on Carlo (MCMC) analyses were conducted with 10,000,000 corrected Akaike Information Criterion (AICc) (Posada 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 Oliveira et al. (2011); Lophiobrycon weitzmani LBP 1225 GU701436 HQ171411 HQ289698 HQ289312 HQ289504 Pereira et al. (2013) Oliveira et al. (2011); Kolpotocheirodontheloura LBP 5033 HM376391 HQ171336 HQ289625 HQ289238 HQ289432 Pereira et al. (2013) Oliveira et al. (2011); Serrapinnus calliurus LBP 3731 HM371155 HQ171291 HQ289580 HQ289195 HQ289388 Pereira et al. (2013)

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

191 Philippine Journal of Science Yu and Quilang: Molecular Phylogeny of Vol. 143 No. 2, December 2014 Philippines Catfishes an acceptance rate of the Metropolis-Hastings MCMC bootstrap support and 1.0 Bayesian posterior probability. sampler ranging between 0.1 and 0.7. The marginal probability, consensus phylograms, and posterior The topology of the trees was the same for ML and probabilities of nodes were estimated from the post-burn- BI (Figure 1). Pangasianodon hypophthalmus of in samples, with 19001 burn-in value for 10,000,000 Pangasiidae, and Plotosidae formed a clade with 90% generations. bootstrap support in the ML and 1.0 Bayesian posterior probability in BI tree. This latter clade is the sister group All DNA sequences generated from this study have been of Ariidae with 100%bootstrap support and 1.0 Bayesian submitted to GenBank (Table 1). 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, RESULTS Ariidae, Plotosidae and Clariidae, is the sister group of Loricariidae. A 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 DISCUSSION sequence were concatenated, totaling to 4216 bp. Data Analysis in Molecular Evolution (DAMBE) version 5.2.57 (Xia 2013) yielded 21 unique sequences from Monophyly of each family 25 concatenated sequences. All 11 species grouped according to their genera and families. Clarias gariepinus, C. macrocephalus, and The concatenated sequences yielded 1526 (36%) variable C. batrachus (Clariidae) formed a monophyletic clade. characters and 1385 (33%) parsimony-informative Clariidae (walking catfishes) is a unique clade by having characters out of 4216 characters. Xia test to determine an arborescent suprabranchial organ (Agnese & Teugels substitution saturation yielded an lss

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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. by having an armored body and a sucker-like ventrally In 2005, Diogo was able to find evidence of monophyly situated mouth (Howes 1983).The monophyly of this of this family based on osteological and myological family was also shown by Cramer et al. (2011) based on characters. Hardman (2005), Betancur-R et al. (2007) and COI, rag1 and rag2. In addition, there are autapomorphies Sullivan et al. (2006) also support the monophyly of this to support the monophyly of this family: these include a family based on molecular data. lack of hyomandibulae fossae, nasal capsule confined to lateral ethmoid, body encased in scutes, and the dilatator The two species of Loricariidae (suckermouth armored opercula muscle is antero-ventrally oriented with presence catfishes), Pterygoplichthys disjunctivus and P. pardalis, of retractor palatine (Howes 1983). formed a monophyletic cluster. Loricariidae is characterized

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Possible synonymy of two Arius species and of two homoplasy resulting in incongruence between molecular Pterygoplichthys species and morphological trees (Diogo 2004). The concatenation of five gene regions was not able to discriminate between Arius manillensis and A. dispar, Loricariidae is separated from the monophyletic and between Pterygoplichthys disjunctivus and P. pardalis clade of Ariidae, Pangasiidae, Clariidae and (Figure 1). This supports previous DNA barcoding studies, Plotosidae which suggest the possibility that the two species of Catfishes are divided into two distinct suborders, the Arius and the two species of Pterygoplichthys are just Loricarioidei and the Siluroidei. The family Loricariidae synonymous (Jumawan et al. 2011; Santos & Quilang belongs to the suborder Loricarioidei, while four 2011; Quilang & Yu 2013). The mean COI genetic other catfish families in this study, namely, Ariidae, divergences between the two Arius species were low Pangasiidae, Clariidae and Plotosidae belong to the in the study of Santos & Quilang (2011) and Quilang suborder Siluroidei (Armbruster 2011). Each of the two & Yu (2013). Low genetic divergence between the two suborders formed a monophyletic clade (Sullivan et al. Pterygoplichthys species was also reported by Wu et al. 2006; Armbruster 2011). The consensus of the studies (2011) and Quilang & Yu (2013) based on cyt b and COI, based on morphology (de Pinna 1998; Diogo 2004) and respectively. The only morphological trait that can be used molecular data (Hardman 2005; Sullivan et al. 2006) is to distinguish between the two Arius species is the pattern that Loricariidae is separated from the clade consisting of tooth patch on the palate: A. manillensis has two large of Ariidae, Pangasiidae, Clariidae and Plotosidae. The ovate tooth patches both on the upper and lower parts of topology of the trees generated in this study (Figure 1) is the palate, while A. dispar has two widely separated tooth consistent with this consensus. patches on the upper palate only (Kailola 1999). On the other hand, vermiculations on the ventral side are used The Loricarioidei was grouped by the derived presence to distinguish between the two Pterygoplichthys species: of odontodes or integumentary teeth (Armbruster P. disjunctivus has continuous and curved dark lines on 2011) and no doubt a monophyletic suborder (Bailey& a white background, whereas P. pardalis has dark spots Baskin 1976; Mo 1991; de Pinna 1998; Hardman 2005; on a white background (Chavez et al. 2006). These traits Sullivan et al. 2006). Howes (1983) diagnosed the six may not be good characters to differentiate the two Arius families (Loricariidae, Astroblepidae, Scoloplacidae, species and the two Pterygoplichthys species (Wu et al. Callichthyidae, Trichomycteridae, Nematogenyidae) 2011; Quilang & Yu 2013). under the Loricarioidei by the presence of encapsulated swimbladder that is divided into separate vesicles, in which Phylogenetic relationships of the five families some part of the cranium contributes to encapsulation. The ML and BI trees generated a (Loricariidae + (Clariidae + (Ariidae + (Pangasiidae + Plotosidae)))) Clariidae is separated from the monophyletic clade topology (Figure 1). This topology is different from the of Ariidae, Pangasiidae and Plotosidae past topology generated by Mo (1991), de Pinna (1998) Analyses show that Clariidae is separated from the and Diogo (2004) based on morphology. Mo’s (1991) monophyletic clade of Ariidae, Pangasiidae and Plotosidae topologies differed on Ariidae, Pangasiidae and Plotosidae (Figure 1). Mo (1991) produced two cladograms based relationship, while de Pinna (1998) and Diogo (2004) on numerical analysis of 126 morphological characters. differed on Clariidae, Ariidae, Pangasiidae and Plotosidae The first cladogram unweighted the 126 morphological relationships. The conflict between the morphological characters, while the second cladogram weighted the 126 and molecular topology can be attributed to homoplasy morphological characters. Both the cladograms of Mo (Davalos et al. 2012). Mo (1991) used 126 morphological (1991) and Hardman (2005) showed that Loricariidae is characters in his analysis and had a Consistency Index more closely related to Clariidae than to the other three (CI) of 0.36. de Pinna’s (1998) work is based on 239 families (Ariidae, Pangasiidae and Plotosidae). 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 CONCLUSION AND inversely proportion to homoplasy index (Diogo 2007). RECOMMENDATIONS These indicate that the phylogenetic topologies based on morphology had higher homoplasy than the phylogenetic Analyses (ML and BI) support the monophyly of each topology generated in this study. Also, catfish is a very of the five catfish families. All topologies showed that diverse group that is highly homoplasic (Diogo 2007). Loricariidae is separated from the monophyletic clade Past morphological studies might neglect to include this formed by the other four catfish families (Ariidae, Clariidae, Pangasiidae, and Plotosidae). Clariidae

194 Philippine Journal of Science Yu and Quilang: Molecular Phylogeny of Vol. 143 No. 2, December 2014 Philippines Catfishes is separated from the monophyletic clade formed Evolution, Chapman & Hall, London, p. 511. by the three catfish families Ariidae, Pangasiidae, BAILEY RM, BASKIN JN. 1976. Scoloplaxdicra, a new and Plotosidae ML and BI analyses gave (Ariidae armored catfish from the Bolivian Amazon. Occas Pap + (Pangasiidae + Plotosidae)) topology. Also, Mus Zool Univ Mich 674:1-14. concatenated sequences of specimens of each of the two species of Arius could not be discriminated from BAKER RH, YU XB, DESALLE R. 1998. Assessing the each other. This is also the case for the two species of relative contribution of molecular and morphological Pterygoplichthys. It is possible that the two species characters in simultaneous analysis trees. Mol Phyl of Arius and the two species of Pterygoplichthys are Evol 9:427-436. synonymous. Future studies may use cytogenomic BAZINET AL, ZWICKL DJ, CUMMINGS MP. 2014. markers such as microsatellites (Pereira et al. 2014) A gateway for phylogenetic analysis powered by grid to establish if the two Arius species and the two computing featuring GARLI 2.0. Syst Biol 63(5):812- Pterygoplichthys species are the same or different 818. species. Future studies may also combine molecular and morphological methods in inferring phylogenetic BETANCUR- R R, ACERO PA, BERMINGHAM E, relationships. The combination of two may improve COOKE R. 2007. Systematics and biogeography of New the partition branch support values (Baker et al. 1998) World sea catfish (Siluriformes: Ariidae) as inferred and remove homoplasy in individual data partitions from mitochondrial, nuclear, and morphological (Farris 1983). evidence. Mol Phylogenet Evol 45(1):339-357. CHAVEZ JM, DELA PAZ RM, MANOHAR SK, PAGULAYAN RC, CARANDANG VI JR. 2006. New Philippine record of South American sailfin ACKNOWLEDGMENTS catfishes(Pisces: Loricariidae). Zootaxa 1109:57–68. We would like to thank the Office of the Chancellor of CONLU PV. 1986. Guide to Philippine Flora and Fauna. the University of the Philippines Diliman, in collaboration Fishes.Vol IX. Manila, Philippines: National Resources with the Office of the Vice Chancellor for Research and Management Center, Ministry of Natural Resources, Development, for funding support through the PhD and the University of the Philippines. 495 p. Incentive Awards (Project No. 111105 PhDIA) given to J. P. Quilang. Thanks also to the Department of Science and CRAMER CA, BONATTO SL, REIS RE. 2011. Technology – Science Education Institute (DOST-SEI) Molecular phylogeny of the Neoplecostominae and Accelerated Science and Technology Human Resource Hypoptopomatinae (Siluriformes: Loricariidae) using Development Program (ASTHRDP) for scholarship and multiple genes. Mol Phylogenet Evol 59:43-52. additional funding support given to Shiny Cathlynne S. Yu. DAVALOS LM, CIRRANELLO AL, GEISLER JH, SIMMONS NB. 2012. Understanding phylogenetic incongruence: lessons from phyllostomid bats. Biol Rev 87:991-1024. REFERENCES DE PINNA MCC. 1998. Phylogenetic relationships of AGNESE JF, TEUGELS GG. 2005. Insight into Neotropical Siluriformes (Teleostei: Ostariophysi): the phylogeny of African Clariidae (Teleostei, historical overview and synthesis of hypotheses. Siluriformes): Implications for their body shape In: Malabarba LR, Reis RE, Vari RP, Lucena CAS, evolution, biogeography, and taxonomy. Mol Lucena ZMS, editors. Phylogeny and Classification of Phylogenet Evol 36:546-553. Neotropical Fishes.Museu de Ciencias e Tecnologia, ALLEN GR. 1998. A review of the marine catfish genus PUCRS, Porto Alegre, Brazil, p. 279-330. Paraplotosus (Plotosidae) with the description of a DIMMICK WW, LARSON A. 1996. A molecular and new species from North-western Australia. Raffles B morphological perspective on the phylogenetic Zool 46(1):123-134. relationships of the otophysan fishes. Mol Phylogenet ARMBRUSTER JW. 2002. Hypancistrus inspector: Evol 1:120-133. A new species of suckermouth armored catfish DIOGO R. 2004. Morphological Evolution, Aptations, (Loricariidae: Ancistrinae). Copeia 1(1):86-92. Homoplasies, Constraints, and Evolutionary Trends: ARMBRUSTER JW. 2011. Global Catfish Biodiversity. Catfishes as a Case Study on General Phylogeny and American Fisheries Society Symposium. 77: 000-001. Macroevolution. Science Publishers, Enfield, USA. AVISE JC. 1994. Molecular Markers, Natural History and DIOGO R. 2005. Morphological evolution, adaptations,

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