Journal of Fish Biology (2009) 74, 377–402 doi:10.1111/j.1095-8649.2008.02077.x, available online at http://www.blackwell-synergy.com

Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes

M. VALDEZ-MORENO*†, N. V. IVANOVA‡, M. ELIAS´ -GUTIERREZ *, S. CONTRERAS-BALDERAS§ AND P. D. N. HEBERT‡ *El Colegio de la Frontera Sur, Av. Centenario km 5.5, Chetumal 77014, Quintana Roo, Mexico, ‡Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, N1G 2W1 Canada and §Bioconservacio´n A. C., A.P. 504, San Nicolas´ de los Garza 66450, Nuevo Leo´n, Mexico

(Received 19 October 2007, Accepted 21 August 2008)

The freshwater fish fauna of Mexico and Guatemala is exceptionally diverse with >600 , many endemic. In this study, patterns of sequence divergence were analysed in representatives of this fauna using cytochrome c oxidase subunit 1 (COI) DNA barcodes for 61 species in 36 genera. The average divergence among conspecific individuals was 045%, while congeneric taxa showed 51% divergence. Three species of , each occupying a different crater lake in the arid regions of Central Mexico, have had a controversial taxonomic history but are usually regarded as endemics to a single lake. They possess identical COI barcodes, suggesting a very recent history of isolation. Representatives of the Cichlidae, a complex and poorly understood family, were well discriminated by barcodes. Many species of Characidae seem to be young, with low divergence values (<2%), but nevertheless, clear barcode clusters were apparent in the Bramocharax–Astyanax complex. The symbranchid, Opisthernon aenigmaticum, has been re- garded as a single species ranging from Guatemala to Mexico, but it includes two deeply divergent barcode lineages, one a possible new endemic species. Aside from these special cases, the results confirm that DNA barcodes will be highly effective in discriminating freshwater fishes from Central America and that a comprehensive analysis will provide new important insights for understanding diversity of this fauna. # 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles

Key words: biodiversity; BOLD; COI; mtDNA; .

INTRODUCTION The fauna of Mexico includes >500 freshwater fish species, almost matching the combined total for the U.S.A. and Canada (Miller, 2005). Another 151 freshwater fishes are known from Guatemala (Froese & Pauly, 2007). High endemism has been recognized in both countries (Miller, 2005; Valdez-Moreno et al., 2005; Froese & Pauly, 2007). Reflecting this diversity, species and generic boundaries in several families are controversial (Miller, 2005). Moreover,

†Author to whom correspondence should be addressed. Tel.: þ52 983 8350440 ext. 4307; fax: þ52 983 8350440 ext. 4321; email: [email protected] 377 # 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles 378 M. VALDEZ-MORENO ET AL. species discovery in this region remains active with at least 13 new species described from Mexico within the past 5 years, including one new of (Rocio) and a new family of catfishes, the Lacantuniidae (Barbour, 2002; Lozano-Vilano, 2002; Minckley et al., 2002; Kallman et al.,2004;Lyons& Mercado-Silva, 2004; Rodiles-Hernandez´ et al., 2005; Strecker, 2005; Schmitter- Soto, 2007). In most cases, understanding of this fish diversity is solely reliant on morphological studies; few taxa have been subjected to molecular analysis (Strecker, 2004; Barriga-Sosa et al., 2005). Mitochondrial sequence diversity has been used to distinguish closely allied species for >20 years (Avise, 1994). More recently, ‘DNA barcoding’, the survey of sequence diversity in a 648 bp segment of the mtDNA gene cytochrome c oxi- dase subunit 1 (COI) has been proposed as a standard tool for species-level iden- tifications of all (Hebert et al., 2003). Aside from the benefits of creating a DNA-based identification system, DNA barcoding is an effective tool for gain- ing an initial sense of the patterning of genetic divergences. Because of this, sev- eral authors have suggested that DNA barcoding will aid rapid progress in traditional taxonomic work by speeding the discovery of new species and in the recognition of synonymies (Gregory, 2005). Although barcoding remains controversial in some circles (Lipscomb et al., 2003; Moritz & Cicero, 2004; Fitzhugh, 2006), it has now been shown to be highly effective in distinguishing species of collembolans (Hogg & Hebert, 2004), ants (Smith et al., 2005), butter- flies (Hebert et al.,2004a), birds (Hebert et al.,2004b) and mammals (Clare et al., 2007; Borisenko et al., 2008). Although fishes represent nearly 50% of all vertebrates, only one large-scale DNA barcoding study has been carried out on this group, and it focused on Australian marine species. This study re- vealed that DNA barcodes were able to identify 100% of the 207 species exam- ined (Ward et al., 2005) and that barcodes were subsequently used to identify marine fish larvae from Australian waters (Pegg et al., 2006; Victor, 2007). The present study builds on this work, by beginning assembly of a DNA bar- code library for the freshwater fishes of Mexico and Guatemala with a focus on species whose taxonomic status has been controversial.

MATERIALS AND METHODS Fishes were collected in 24 diverse freshwater environments from Mexico and Guatemala, ranging from southern tropical forests to semi-deserts in the north (Fig. 1). Details on collect- ing localities, co-ordinates and dates are available within the project file ‘Freshwater Fishes of Mexico’ on the Barcode of Life Data System (BOLD) (Ratnasingham & Hebert, 2007). A small piece (1–3 mm3) of muscle was removed from the left side of each fish and placed in 100% ethanol using tools that were flame sterilized before sampling each specimen. The remainder of each fish is preserved as a reference voucher in the Fish Collection of El Colegio de la Frontera Sur, Chetumal Unit (ECOCHP), Escuela Na- cional de Ciencias Biologicas´ (ENCB, IPN) and the Universidad Autonoma´ de Nuevo Leon´ (Monterrey, Mexico), and accession numbers are included in BOLD. Whenever possible, at least five adults of each species were sampled. In the case of difficult taxa, more specimens were obtained. All identifications were based on specialized literature and consultation with taxonomic specialists in cases when the identification was partic- ularly difficult. Names follow FishBase (Froese & Pauly, 2007). DNA barcoding was carried out at the Canadian Centre for DNA Barcoding using standard protocols (Hajibabaei et al., 2005). DNA was extracted from 1 mm3 tissue plugs

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FIG. 1. Approximate locations of all collection sites in Mexico and Guatemala for freshwater fish barcoded. Additionally, the following marks denote positive localities for: , Astyanax mexicanus; , Astyanax aeneus; , Bramocharax caballeroi, only found in Catemaco lake; , Bramocharax baileyi; , Poblana species. that were sub-sampled into a vertebrate lysis buffer with proteinase K and digested over- night at 56° C. Genomic DNA was subsequently extracted using a membrane-based approach on a Biomek NXÒ (www.pall.com) liquid handling station using AcroPrep 96 (www.beckman.com), 1 ml filter plates with 10 mm PALL glass fibre media (Ivanova et al., 2006). A 652–658 bp segment of COI was amplified using different combinations of fish primers: FishF1, FishR1, FishF2, FishR2 (Ward et al., 2005) or the M13-tailed primer cock- tails C_Fish F1t1 – C_FishR1t1 and C_VF1LFt1 – C_VR1LRt1 (Ivanova et al., 2007). The 125 ml polymerase chain reaction (PCR) mixes included 625 ml 10% trehalose, 2 ml ultrapure water, 125 ml10 PCR buffer, 0625 MgCl2 (50 mM), 0125 ml of each primer (001 mM), 00625 ml of each dNTP (005 mM), 0625 ml Taq polymerase (www.net.com) and 20 ml DNA template. Amplification protocols followed those described in earlier publications (Hajibabaei et al., 2005). PCR products were visualized on pre-cast agarose gels (E-GelsÒ; www.invitrogen.com), and the most intense products were selected for sequencing. Products were labelled with the BigDyeÒ Terminator v.3.1 Cycle Sequencing Kit (www.appliedbiosystems.com) using standard methods (Hajibabaei et al., 2005) and sequenced bidirectionally using an ABI 3730 capillary sequencer. Sequences were edited and aligned using SEQSCAPE v.2.1.1 (Applied Biosystems Inc.). Sequence divergences were calculated using the Kimura two-parameter (K2P) distance model (Kimura, 1980). Sequence data, electropherograms, trace files, primer details, photo- graphs and collection localities for specimens are available within the project file on BOLD (http://www.barcodinglife.org; see further information). Sequences have also been deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank). All accession num- bers in both databases are included in Appendix I. Neighbour-joining (NJ) trees of K2P distances were created to provide a graphical representation of the patterning of divergence between species (Saitou & Nei, 1987).

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A simplified ID tree of all the species was generated with MEGA 3 software (Kumar et al., 2004).

RESULTS In total, 427 fishes were sequenced including 61 species in 36 genera and 15 families, representing 10% of the known fauna. Accession numbers to BOLD and GenBank sequences for each specimen are provided in Appendix I. Read lengths were all over 600 bp long, and no insertions, deletions or stop codons were observed. The full K2P–NJ tree is provided in Appendix II, but Fig. 2 provides an overview of the patterning of sequence divergences. The average K2P distance among conspecific individuals for all species was 045% compared with 51% for species within the genera (Table I). Overall, 93% of nominal species recognized by prior taxonomic work were dis- criminated by barcodes. The most conspicuous case of failed resolution involved three species of Poblana (Atherinopsidae). Although other members of this family were correctly discriminated by the barcodes, the three Poblana species were included in the same barcode cluster. As noted later, the failure to discriminate these species may reflect biological reality that these are members of a single species. Among the Characidae, members of two genera (Astyanax and Bramocharax) were difficult to discriminate because they showed low divergences (<31%) between species and genera. Nevertheless, detailed analyses of the ID tree (Fig. 2) for the Astyanax–Bramocharax complex demonstrated a consistent pattern of biogeographic associations. Specimens of Astyanax mexicanus (De Filippi) from northern populations formed a barcode cluster separate from a second group consisting of Astyanax aeneus (Gunther),¨ restricted to the humid tropical southeast and Central America. A third cluster was formed by Bramocharax caballeroi Contreras-Balderas & Rivera-Teillery representing the endemic population in Catemaco Lake, Veracruz, a locality distant from Central America, where all other species of this genus occur. A fourth barcode group included specimens of Bramocharax baileyi Rosen (MX649 and 652 in BOLD) together with 10 juvenile forms identified by their matching barcodes. All 16 species were well discriminated by barcodes, and members of this family themselves formed a compact, well-defined cluster. Barcode results showed that Ophisternon aenigmaticum Rosen & Greenwood, from the Catemaco Lake (Mexico) and Chisec River (Guatemala) had very high divergences of 9%, suggesting that they might be separate species, a conclusion that requires further taxonomic investigation. All members of the other 10 families (Poeciliidae, Cyprinodontidae, Clupeidae, Centrarchidae, Catostomidae, Cyprinidae, Ariidae, Ictaluridae, Rivulidae and Pimelodidae) were well discriminated by the barcodes. Only three species (Cyprinodon artifrons Hubbs, Garmanella pulchra Hubbs, and Brycon guatemalensis Regan) fell outside their family cluster, but they were well discriminated.

DISCUSSION Congeneric divergences were c. 12 greater than those between conspecifics, a ratio slightly lower than that (25) reported in an earlier study on Australian

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FIG. 2. Neighbour-joining tree of 428 cytochrome c oxidase subunit 1 sequences (including menidia from GenBank) for 61 identified freshwater fish species using Kimura two-parameter distances. The number of specimens sequenced is in brackets. Specimen details are available in Barcode of Life Data System (http://www.barcodinglife.org/).

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 382 M. VALDEZ-MORENO ET AL.

TABLE I. Genetic divergences (Kimura two-parameter) at different taxonomic levels for freshwater fish. *Because of Poblana species, see text

Comparisons Number of Minimum Distance Maximum within comparisons (%) mean (%) (%) S.E.

Species 3399 0 045 211 001 Genera 1699 0* 510 1667 008 Families 10 286 1084 1357 2848 007 Orders 3087 1724 2338 2877 003 Classes 72 881 1788 2534 3116 001

fishes (Ward et al., 2005). Perhaps, freshwater species are on average more recent in origin than their marine counterparts.

THE ATHERINOPSIDAE CLUSTER The family Atherinopsidae includes several important species of silversides much appreciated as food in Central Mexico (Barriga-Sosa et al., 2005). Unfor- tunately, most of their fisheries are becoming depleted because of introduced species and overfishing (Lyons et al., 1998). The taxonomy of the group is controversial. The genus Poblana itself is often placed within Menidia and the most recent revision regarded its three species as sub-species within Menidia alchichica de Buen (Echelle & Echelle, 1984; Miller, 2005). However, the three Poblana species (Poblana alchichica de Buen, Poblana letholepis Alvarez, and Poblana squamata Alvarez) are still recognized by FishBase (Froese & Pauly, 2007). The barcode congruence that the authors observed among these three taxa supports the conclusion that the three taxa of Poblana are conspecific. A comparison including the sequences from these three nominal sequences of Poblana show just 8% divergence from a GenBank record for Menidia menidia (L.), a divergence value common for congeneric taxa. The other atherinopsid names used in this study are accepted by most ichthyologists, but discussion about the status of these genera will continue. All other atherinopsids considered in this study ( jordani Woolman, Chirostoma riojai Solorzano´ & Lopez,´ and Chirostoma labarcae Meek) were well discriminated by the barcodes.

CHARACIDAE: THE CASE OF ASTYANAX AND BRAMOCHARAX Barcode data failed to separate the genera Bramocharax and Astyanax, conflict- ing with the views of most ichthyologists (Reis et al., 2003; Valdez-Moreno et al., 2005). However, it has been recognized that the juvenile stages of these two genera are very similar. For example, immature specimens of B. baileyi have a dentition and general morphology closely resembling that typical of Astyanax. All the species named in this study are considered valid by specialists (Contreras- Balderas & Rivera-Teillery, 1985; Reis et al., 2003; Miller, 2005; Valdez-Moreno et al., 2005) because of their morphological differences.

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Although it is not possible to solve this systematic problem, without further investigation barcode, studies can reveal situations in need of more detailed analysis and potential taxonomic revisions. Thirty years after their description (Rosen, 1970, 1972), most Bramocharax species have only been re–collected once (Valdez-Moreno et al., 2005). The specimens barcoded in this study were collected from their type localities, intermittent rivers in the north of Guatemala, where they are rare endemics. The only well-discriminated characid was B. guatemalensis.

THE CICHLIDAE, ONE OF THE MOST DIVERSE FAMILIES OF FRESHWATER FISHES Cichlids are a complex family with >1000 species distributed in the tropics (Berra, 2001). The 100þ species in Central America comprise a taxonomical puzzle at both species and generic levels (Miller, 2005). For example, in a single Nicaraguan Lake, four controversial sympatric species have recently been dis- covered (Barluenga & Meyer, 2004; Barluenga et al., 2006; Gavrilets et al., 2007; Stauffer et al., 2008). Unlike the Characide, the cichlids are considered an old group that has secondarily invaded freshwater habitats (Miller, 2005). The results on 16 cichlid species indicate that barcodes can significantly advance understanding of the diversity of this group, at least in Meso-America. However, in the case of species with recent evolution, the use of this single gene should be augmented with different approaches, as the case of Amphilophus from Lake Apoyo (Nicaragua) (Barluenga et al., 2006). The authors expect that DNA barcodes will become a strategic tool to the understanding of the distribution of species, as exemplified by the discovery of maculicauda (Regan) in Lachua´ Lake representing a range extension of this species to Alta Verapaz, Guatemala (Valdez-Moreno et al., 2005). The work also revealed the establishment of two exotic African cichlids, Oreochromis niloticus (L.) and Oreochromis mossambicus (Peters), in Mexico. Their presence was rec- ognized by the barcode matching of immature stages with sequence records in BOLD (Ratnasingham & Hebert, 2007) as the latter two species form a cluster clearly distinct from native American species. One specimen of Thorichthys (MX542), was also a juvenile lacking the morphological diagnostic features in this genus.

THE DISCOVERY OF NEW SPECIES, THE CASE OF SYNBRANCHIDAE All swamp eels are mainly assigned to a genus on the basis of their gill openings. All specimens with crescent transverse slits are placed in the genus Ophisternon represented in this region by two species: the eyeless Ophisternon infernale (Hubbs) and the eyed O. aenigmaticum. The latter species is distributed from northeast South America to the Mexican Gulf coast with records in hypersaline waters from Belize (Miller, 2005). The Chisec material barcoded in this study is likely true O. aenigmaticum because this species was described from a nearby site (Sebol River). If so, the specimens from Catemaco Lake probably represent a new species in this genus, providing an example of the way in which barcodes can aid the discovery of specimens meriting more

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 384 M. VALDEZ-MORENO ET AL. intensive taxonomic study. Ward et al. (2008) report another example of a pos- sible new fish species uncovered by DNA barcodes involving a key commercial species – the barramundi, Lates calcarifer (Bloch). Although ID trees based on COI sequence divergences are not primarily a phylogenetic tool, they do provide some signal of deeper relationships as noted by Ward et al. (2005). This result was reinforced in this study as species in a genus and genera in a family generally formed cohesive clusters. Although COI is not an adequate tool to build a fish phylogeny, the authors do expect that insights on relationships will grow as taxon coverage expands. There was one partial specimen, regarded as a Cyprinidae from Peten Lake. Although it was not possible to identify it, the barcode record will allow its future identification once a barcode record that matches it is obtained from an adult voucher. This capacity to identify fragmentary specimens will be another valuable attribute of barcodes. This study reinforces and extends conclusions from prior DNA barcode studies on fishes. Barcodes recognized 93% of the freshwater fish species included in the study, and most cases of incomplete resolution involved species of Poblana, which likely reflect a case of over-splitting. As such, barcodes deliv- ered an identification for some 98% of the species in this study. Aside from their role as an effective ID instrument, barcodes can also help in recognizing groups and sub-groups below the family level, especially in lineages with long histories of evolutionary divergence. Barcoding will be less precise in delivering identifications in very young species groups, but these are rare. The authors emphasize that the construction of an effective barcode reference library is strictly dependent on access to reliably identified specimens. The authors also expect that barcoding will be a great aid in the recognition of ID mistakes, syn- onyms, larvae–adult associations and in the identification of semi-digested items in food studies. The authors close by emphasizing that all deeply divergent bar- code lineages should be exposed to standard biosystematics investigations.

FURTHER INFORMATION Further information is included in the project ‘Freshwater Fishes of Mexico’ on BOLD (http://www.barcodinglife.org/) and in Appendixes I and II.

We thank D. Cazarez, C. Q. Lizama and J. J. S. Soto for aiding with field collections and identifications. R. Herrera also assisted in field collections, while most specimen photographs were taken by H. B. Basave, who also participated in fieldwork. This pro- ject was carried out during a sabbatical leave of M. Valdez-Moreno and M. Elıas-´ Gutierrez at the Department of Integrative Biology, University of Guelph. All members of the Hebert Laboratory, particularly T. Zemlak, D. Steinke and G. Downs, provided helpful discussions about molecular techniques and bioinformatics. The Centro de Es- tudios Conservacionistas, particularly its director, Jorge Ruiz Ordon˜ez, and the Com- ision´ Nacional de Areas Protegidas de Guatemala, particularly F. Herrera and E. Secaira, aided the acquisition of collection permits for Guatemala, including several protected areas. M. L. Vilano and M. E. G. Ramırez´ provided access to material in the ichthyological collection of the Universidad Autonoma´ de Nuevo Leon.´ F. M. Jeronimo´ and G. F. Lucero from the Instituto Politecnico Nacional (Mexico) kindly donated identified specimens of Atherinopsidae family. We also thank R. Ward for his valuable editorial suggestions to an earlier draft of this manuscript.

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Smith, M. A., Fisher, B. L. & Hebert, P. D. N. (2005). DNA barcoding for effective biodiversity assessment of a hyperdiverse arthropod group: the ants of Madagas- car. Philosophical Transactions of the Royal Society B 360, 1825–1834. Stauffer, J. R., McCrary, J. K. & Black, K. E. (2008). Three new species of cichlid fishes (Teleostei: Cichlidae) from Lake Apoyo, Nicaragua. Proceedings of the Biological Society of Washington 121, 117–129. Strecker, U. (2004). The Cyprinodon species flock from Laguna Chichancanab, Mexico (Teleostei): sexual and disruptive selection driving adaptive radiation. Mitteilungen aus dem Hamburgischen Zoologischen Museum und Institut 101, 65–74. Strecker, U. (2005). Description of a new species from Laguna Chichancanab, Yucatan, Mexico: Cyprinodon suavium (Pisces: Cyprinodontidae). Hydrobiologia 541, 107– 115. Valdez-Moreno, M., Pool-Canul, J. & Contreras-Balderas, S. (2005). A checklist of the freshwater ichthyofauna from El Peten and AltaVerapaz, Guatemala, with notes for its conservation and management. Zootaxa 1072, 43–60. Victor, B. C. (2007). Coryphopterus kuna, a new goby (Perciformes: Gobiidae: Gobiinae) from the western Caribbean, with the identification of the late larval stage and an estimate of the pelagic larval duration. Zootaxa 1526, 51–61. Ward, R. D., Zemlak, T. S., Innes, B. H., Last, P. R. & Hebert, P. D. N. (2005). DNA barcoding Australia’s fish species. Philosophical Transactions of the Royal Society B, 360, 1847–1857. doi: 10.1098/rstb.2005.1716 Ward, R. D., Holmes, B. H. & Yearsley, G. K. (2008). DNA barcoding reveals a likely second species of Asian seabass (barramundi) Lates calcarifer. Journal of Fish Biology 72, 458–463. doi: 10.1111/j.1095-8649.2007.01703.x

Electronic Reference Froese, R. & Pauly, D. (2007). FishBase. Available at http://www.fishbase.org August 28, 2007.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 388 M. VALDEZ-MORENO ET AL.

APPENDIX I. Accession numbers in Barcode of Life Data System (BOLD) and GenBank for all specimens included in this study

Species BOLD process ID GenBank ID

Astyanax aeneus MEFM140-05 EU751625 A. aeneus MEFM141-05 EU751626 A. aeneus MEFM142-05 EU751627 A. aeneus MEFM139-05 EU751628 Astyanax mexicanus MEFM499-06 EU751629 A. mexicanus MEFM503-06 EU751630 A. mexicanus MEFM502-06 EU751631 A. mexicanus MEFM504-06 EU751632 A. mexicanus MEFM498-06 EU751633 A. mexicanus MEFM240-05 EU751634 A. mexicanus MEFM241-05 EU751635 A. mexicanus MEFM235-05 EU751636 A. mexicanus MEFM236-05 EU751637 A. mexicanus MEFM237-05 EU751638 A. mexicanus MEFM232-05 EU751639 A. mexicanus MEFM239-05 EU751640 A. mexicanus MEFM501-06 EU751641 A. mexicanus MEFM233-05 EU751642 A. mexicanus MEFM205-05 EU751643 A. mexicanus MEFM204-05 EU751644 A. mexicanus MEFM203-05 EU751645 A. mexicanus MEFM206-05 EU751646 A. mexicanus MEFM198-05 EU751647 A. mexicanus MEFM238-05 EU751648 A. mexicanus MEFM201-05 EU751649 A. mexicanus MEFM200-05 EU751650 A. mexicanus MEFM197-05 EU751651 A. mexicanus MEFM202-05 EU751652 A. mexicanus MEFM234-05 EU751653 A. mexicanus MEFM500-06 EU751654 A. mexicanus MEFM199-05 EU751655 A. mexicanus MEFM505-06 EU751656 A. mexicanus MEFM496-06 EU751657 A. mexicanus MEFM497-06 EU751658 A. mexicanus MEFM495-06 EU751659 A. mexicanus MEFM494-06 EU751660 A. mexicanus MEFM493-06 EU751661 sp.1 MEFM575-06 EU751662 Belonesox belizanus MEFM180-05 EU751663 B. belizanus MEFM181-05 EU751664 B. belizanus MEFM177-05 EU751665 B. belizanus MEFM178-05 EU751666 B. belizanus MEFM179-05 EU751667 Bramocharax baileyi MEFM654-06 EU751668 B. baileyi MEFM617-06 EU751669 B. baileyi MEFM649-06 EU751670

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 FRESHWATER FISH BARCODES 389

APPENDIX I. Continued

Species BOLD process ID GenBank ID

B. baileyi MEFM652-06 EU751671 B. baileyi MEFM650-06 EU751672 B. baileyi MEFM651-06 EU751673 B. baileyi MEFM653-06 EU751674 B. baileyi MEFM518-06 EU751675 B. baileyi MEFM519-06 EU751676 B. baileyi MEFM520-06 EU751677 B. baileyi MEFM521-06 EU751678 B. baileyi MEFM522-06 EU751679 Bramocharax caballeroi MXII019-07 EU751680 B. caballeroi MXII020-07 EU751681 B. caballeroi MXII021-07 EU751682 B. caballeroi MXII022-07 EU751683 B. caballeroi MXII023-07 EU751684 B. caballeroi MXII024-07 EU751685 B. caballeroi MXII025-07 EU751686 B. caballeroi MXII026-07 EU751687 B. caballeroi MXII027-07 EU751688 B. caballeroi MXII028-07 EU751689 B. caballeroi MXII029-07 EU751690 B. caballeroi MXII030-07 EU751691 B. caballeroi MXII031-07 EU751692 B. caballeroi MXII032-07 EU751693 B. caballeroi MXII033-07 EU751694 B. caballeroi MXII034-07 EU751695 B. caballeroi MXII035-07 EU751696 B. caballeroi MXII036-07 EU751697 B. caballeroi MXII037-07 EU751698 B. caballeroi MXII038-07 EU751699 B. caballeroi MXII039-07 EU751700 B. caballeroi MXII040-07 EU751701 B. caballeroi MXII041-07 EU751702 B. caballeroi MXII042-07 EU751703 B. caballeroi MXII043-07 EU751704 B. caballeroi MXII044-07 EU751705 B. caballeroi MXII045-07 EU751706 B. caballeroi MXII046-07 EU751707 B. caballeroi MXII047-07 EU751708 B. caballeroi MXII048-07 EU751709 B. caballeroi MXII049-07 EU751710 B. caballeroi MXII050-07 EU751711 B. caballeroi MXII051-07 EU751712 B. caballeroi MXII052-07 EU751713 B. caballeroi MXII053-07 EU751714 B. caballeroi MXII054-07 EU751715 B. caballeroi MXII055-07 EU751716 B. caballeroi MXII056-07 EU751717

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 390 M. VALDEZ-MORENO ET AL.

APPENDIX I. Continued

Species BOLD process ID GenBank ID

B. caballeroi MXII057-07 EU751718 B. caballeroi MXII058-07 EU751719 B. caballeroi MXII059-07 EU751720 B. caballeroi MEFM051-05 EU751721 B. caballeroi MEFM055-05 EU751722 B. caballeroi MEFM054-05 EU751723 B. caballeroi MEFM052-05 EU751724 B. caballeroi MEFM053-05 EU751725 Brycon guatemalensis MEFM576-06 EU751726 B. guatemalensis MEFM577-06 EU751727 B. guatemalensis MEFM578-06 EU751728 B. guatemalensis MEFM579-06 EU751729 B. guatemalensis MEFM580-06 EU751730 Campostoma anomalum MEFM491-06 EU751731 C. anomalum MEFM492-06 EU751732 C. anomalum MEFM506-06 EU751733 Chirostoma jordani MEFM729-06 EU751734 C. jordani MEFM728-06 EU751735 C. jordani MEFM727-06 EU751736 C. jordani MEFM726-06 EU751737 C. jordani MEFM712-06 EU751738 C. jordani MEFM711-06 EU751739 C. jordani MEFM710-06 EU751740 C. jordani MEFM709-06 EU751741 C. jordani MEFM708-06 EU751742 Chirostoma labarcae MEFM725-06 EU751743 C. labarcae MEFM724-06 EU751744 C. labarcae MEFM723-06 EU751745 C. labarcae MEFM722-06 EU751746 Chirostoma riojai MEFM735-06 EU751747 C. riojai MEFM734-06 EU751748 C. riojai MEFM733-06 EU751749 C. riojai MEFM732-06 EU751750 C. riojai MEFM731-06 EU751751 Cichlasoma salvini MEFM572-06 EU751757 Cichlasoma urophthalmus MEFM812-06 EU751758 C. urophthalmus MEFM813-06 EU751759 C. urophthalmus MEFM814-06 EU751760 C. urophthalmus MEFM815-06 EU751761 C. urophthalmus MEFM816-06 EU751762 C. urophthalmus MEFM645-06 EU751763 C. urophthalmus MEFM646-06 EU751764 Cyprinella lutrensis MEFM485-06 EU751765 C. lutrensis MEFM484-06 EU751766 C. lutrensis MEFM481-06 EU751767 C. lutrensis MEFM482-06 EU751768 C. lutrensis MEFM483-06 EU751769

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 FRESHWATER FISH BARCODES 391

APPENDIX I. Continued

Species BOLD process ID GenBank ID

Cyprinella rutila MEFM218-05 EU751770 C. rutila MEFM221-05 EU751771 C. rutila MEFM220-05 EU751772 C. rutila MEFM219-05 EU751773 C. rutila MEFM217-05 EU751774 Cyprinidae MEFM658-06 EU751775 Cyprinodon artifrons MEFM416-06 EU751776 C. artifrons MEFM468-06 EU751777 C. artifrons MEFM412-06 EU751778 C. artifrons MEFM469-06 EU751779 C. artifrons MEFM409-06 EU751780 C. artifrons MEFM467-06 EU751781 C. artifrons MEFM414-06 EU751782 C. artifrons MEFM415-06 EU751783 C. artifrons MEFM413-06 EU751784 C. artifrons MEFM411-06 EU751785 C. artifrons MEFM410-06 EU751786 C. artifrons MEFM418-06 EU751787 C. artifrons MEFM417-06 EU751788 Dionda melanops MEFM216-05 EU751789 D. melanops MEFM215-05 EU751790 D. melanops MEFM214-05 EU751791 D. melanops MEFM212-05 EU751792 D. melanops MEFM213-05 EU751793 Dorosoma petenense MEFM042-05 EU751794 D. petenense MEFM045-05 EU751795 D. petenense MEFM479-06 EU751796 D. petenense MEFM044-05 EU751797 D. petenense MEFM043-05 EU751798 D. petenense MEFM477-06 EU751799 D. petenense MEFM041-05 EU751800 D. petenense MEFM476-06 EU751801 D. petenense MEFM475-06 EU751802 D. petenense MEFM478-06 EU751803 Fundulus cf. grandis MEFM489-06 EU751804 F. cf. grandis MEFM490-06 EU751805 F. cf. grandis MEFM487-06 EU751806 F. cf. grandis MEFM488-06 EU751807 F. cf. grandis MEFM486-06 EU751808 Gambusia sexradiata MEFM008-05 EU751809 G. sexradiata MEFM009-05 EU751810 Gambusia yucatana MEFM006-05 EU751811 G. yucatana MEFM007-05 EU751812 G. yucatana MEFM001-05 EU751813 G. yucatana MEFM255-06 EU751814 G. yucatana MEFM254-06 EU751815 G. yucatana MEFM253-06 EU751816

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 392 M. VALDEZ-MORENO ET AL.

APPENDIX I. Continued

Species BOLD process ID GenBank ID

G. yucatana MEFM256-06 EU751817 G. yucatana MEFM252-06 EU751818 Garmanella pulchra MEFM405-06 EU751819 G. pulchra MEFM408-06 EU751820 G. pulchra MEFM406-06 EU751821 G. pulchra MEFM407-06 EU751822 G. pulchra MEFM404-06 EU751823 Heterandria bimaculata MEFM614-06 EU751824 H. bimaculata MEFM615-06 EU751825 H. bimaculata MEFM523-06 EU751826 H. bimaculata MEFM525-06 EU751827 H. bimaculata MEFM526-06 EU751828 H. bimaculata MEFM595-06 EU751829 H. bimaculata MEFM596-06 EU751830 H. bimaculata MEFM597-06 EU751831 H. bimaculata MEFM598-06 EU751832 H. bimaculata MEFM599-06 EU751833 H. bimaculata MEFM567-06 EU751834 H. bimaculata MEFM568-06 EU751835 H. bimaculata MEFM569-06 EU751836 H. bimaculata MEFM570-06 EU751837 H. bimaculata MEFM571-06 EU751838 Hexanematichthys assimilis MEFM156-05 EU751839 H. assimilis MEFM158-05 EU751840 H. assimilis MEFM157-05 EU751841 H. assimilis MEFM155-05 EU751842 H. assimilis MEFM154-05 EU751843 Ictalurus furcatus MEFM657-06 EU751844 Lepomis megalotis MEFM208-05 EU751845 L. megalotis MEFM207-05 EU751846 L. megalotis MEFM209-05 EU751847 L. megalotis MEFM211-05 EU751848 L. megalotis MEFM210-05 EU751849 Menidia menidia FRFM001-07 EU751850 Moxostoma albidum MEFM231-05 EU751965 M. albidum MEFM228-05 EU751966 M. albidum MEFM229-05 EU751967 M. albidum MEFM230-05 EU751968 M. albidum MEFM227-05 EU751969 Notropis amabilis MEFM224-05 EU751851 N. amabilis MEFM222-05 EU751852 N. amabilis MEFM223-05 EU751853 Notropis cf. stramineus MEFM226-05 EU751854 N. cf. stramineus MEFM225-05 EU751855 Notropis chihuahua MEFM508-06 EU751856 N. chihuahua MEFM509-06 EU751857 N. chihuahua MEFM507-06 EU751858

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 FRESHWATER FISH BARCODES 393

APPENDIX I. Continued

Species BOLD process ID GenBank ID

Ophisternon aenigmaticum MEFM616-06 EU751859 O. aenigmaticum MEFM626-06 EU751860 O. aenigmaticum MEFM627-06 EU751861 O. aenigmaticum MEFM630-06 EU751862 Ophisternon sp. 1 MXII008-07 EU751863 Ophisternon sp. 1 MXII009-07 EU751864 Ophisternon sp. 1 MXII010-07 EU751865 Ophisternon sp. 1 MXII011-07 EU751866 Ophisternon sp. 1 MXII012-07 EU751867 Ophisternon sp. 1 MXII013-07 EU751868 Ophisternon sp. 1 MXII014-07 EU751869 Ophisternon sp. 1 MXII015-07 EU751870 Ophisternon sp. 1 MXII016-07 EU751871 Ophisternon sp. 1 MXII017-07 EU751872 Ophisternon sp. 1 MXII018-07 EU751873 Ophisternon sp. 1 MEFM058-05 EU751874 Ophisternon sp. 1 MEFM060-05 EU751875 Ophisternon sp. 1 MEFM056-05 EU751876 Ophisternon sp. 1 MEFM059-05 EU751877 Ophisternon sp. 1 MEFM057-05 EU751878 Oreochromis mossambicus MEFM196-05 EU751879 Oreochromis niloticus MEFM195-05 EU751880 O. niloticus MEFM192-05 EU751881 O. niloticus MEFM194-05 EU751882 O. niloticus MEFM193-05 EU751883 Parachromis friedrichsthalii MEFM186-05 EU751884 P. friedrichsthalii MEFM182-05 EU751885 P. friedrichsthalii MEFM183-05 EU751886 P. friedrichsthalii MEFM185-05 EU751887 P. friedrichsthalii MEFM184-05 EU751888 P. friedrichsthalii MEFM581-06 EU751889 P. friedrichsthalii MEFM582-06 EU751890 P. friedrichsthalii MEFM583-06 EU751891 P. friedrichsthalii MEFM584-06 EU751892 P. friedrichsthalii MEFM585-06 EU751893 P. friedrichsthalii MEFM618-06 EU751894 P. friedrichsthalii MEFM619-06 EU751895 P. friedrichsthalii MEFM620-06 EU751896 P. friedrichsthalii MEFM621-06 EU751897 P. friedrichsthalii MEFM622-06 EU751898 Petenia splendida MEFM641-06 EU751899 P. splendida MEFM642-06 EU751900 P. splendida MEFM643-06 EU751901 Poblana alchichica MEFM742-06 EU751902 Poblana letholepis MEFM741-06 EU751903 P. letholepis MEFM740-06 EU751904 Poblana squamata MEFM739-06 EU751905

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 394 M. VALDEZ-MORENO ET AL.

APPENDIX I. Continued

Species BOLD process ID GenBank ID

P. squamata MEFM738-06 EU751906 Poecilia mexicana MEFM854-06 EU751907 P. mexicana MEFM855-06 EU751908 P. mexicana MEFM856-06 EU751909 P. mexicana MEFM857-06 EU751910 P. mexicana MEFM243-06 EU751911 P. mexicana MEFM242-06 EU751912 P. mexicana MEFM246-06 EU751913 P. mexicana MEFM245-06 EU751914 P. mexicana MEFM244-06 EU751915 P. mexicana MEFM010-05 EU751916 P. mexicana MEFM011-05 EU751917 P. mexicana MEFM013-05 EU751918 P. mexicana MEFM012-05 EU751919 P. mexicana MEFM002-05 EU751920 P. mexicana MEFM574-06 EU751921 P. mexicana MEFM594-06 EU751922 P. mexicana MEFM612-06 EU751923 P. mexicana MEFM613-06 EU751924 P. mexicana MEFM631-06 EU751925 P. mexicana MEFM632-06 EU751926 P. mexicana MEFM633-06 EU751927 P. mexicana MEFM634-06 EU751928 P. mexicana MEFM635-06 EU751929 P. mexicana MEFM573-06 EU751930 P. mexicana MEFM524-06 EU751931 P. mexicana MEFM527-06 EU751932 P. mexicana MEFM591-06 EU751933 P. mexicana MEFM592-06 EU751934 P. mexicana MEFM593-06 EU751935 P. mexicana MEFM636-06 EU751936 P. mexicana MEFM637-06 EU751937 P. mexicana MEFM638-06 EU751938 P. mexicana MEFM639-06 EU751939 P. mexicana MEFM640-06 EU751940 Poecilia petenensis MEFM853-06 EU751941 Poeciliopsis catemaco MEFM038-05 EU751942 P. catemaco MEFM036-05 EU751943 P. catemaco MEFM040-05 EU751944 P. catemaco MEFM037-05 EU751945 P. catemaco MEFM039-05 EU751946 Poeciliopsis pleurospilus MEFM510-06 EU751947 P. pleurospilus MEFM512-06 EU751948 P. pleurospilus MEFM513-06 EU751949 P. pleurospilus MEFM514-06 EU751950 P. pleurospilus MEFM511-06 EU751951 Potamarius nelsoni MEFM656-06 EU751952

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 FRESHWATER FISH BARCODES 395

APPENDIX I. Continued

Species BOLD process ID GenBank ID

Rocio octofasciata MEFM016-05 EU751752 R. octofasciata MEFM015-05 EU751753 R. octofasciata MEFM017-05 EU751754 R. octofasciata MEFM014-05 EU751755 R. octofasciata MEFM003-05 EU751756 Rhamdia guatemalensis MEFM474-06 EU751953 R. guatemalensis MEFM473-06 EU751954 R. guatemalensis MEFM470-06 EU751955 R. guatemalensis MEFM472-06 EU751956 R. guatemalensis MEFM471-06 EU751957 R. guatemalensis MEFM655-06 EU751958 R. guatemalensis MEFM528-06 EU751959 R. guatemalensis MEFM529-06 EU751960 R. guatemalensis MEFM530-06 EU751961 R. guatemalensis MEFM531-06 EU751962 Rhamdia sp. 1 MEFM532-06 EU751963 Rivulus tenuis MEFM611-06 EU751964 Theraps lentiginosus MEFM548-06 EU751970 T. lentiginosus MEFM549-06 EU751971 Thorichthys helleri MEFM543-06 EU751972 T. helleri MEFM544-06 EU751973 T. helleri MEFM545-06 EU751974 T. helleri MEFM546-06 EU751975 Thorichthys meeki MXII383-07 EU751976 T. meeki MXII384-07 EU751977 T. meeki MXII385-07 EU751978 T. meeki MXII386-07 EU751979 T. meeki MXII387-07 EU751980 T. meeki MXII389-07 EU751981 T. meeki MXII390-07 EU751982 T. meeki MXII391-07 EU751983 T. meeki MXII392-07 EU751984 T. meeki MEFM021-05 EU751985 T. meeki MEFM020-05 EU751986 T. meeki MEFM019-05 EU751987 T. meeki MEFM018-05 EU751988 T. meeki MEFM004-05 EU751989 T. meeki MEFM586-06 EU751990 T. meeki MEFM587-06 EU751991 T. meeki MEFM588-06 EU751992 T. meeki MEFM589-06 EU751993 T. meeki MEFM590-06 EU751994 T. meeki MEFM538-06 EU751995 T. meeki MEFM539-06 EU751996 T. meeki MEFM540-06 EU751997 T. meeki MEFM541-06 EU751998 T. meeki MEFM547-06 EU751999

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 396 M. VALDEZ-MORENO ET AL.

APPENDIX I. Continued

Species BOLD process ID GenBank ID

Thorichthys sp. 1 MEFM542-06 EU752000 Vieja fenestrata MEFM049-05 EU752001 V. fenestrata MEFM050-05 EU752002 V. fenestrata MEFM047-05 EU752003 V. fenestrata MEFM048-05 EU752004 V. fenestrata MEFM046-05 EU752005 Vieja godmanni MEFM557-06 EU752006 V. godmanni MEFM558-06 EU752007 V. godmanni MEFM559-06 EU752008 V. godmanni MEFM560-06 EU752009 V. godmanni MEFM561-06 EU752010 Vieja intermedia MEFM600-06 EU752011 V. intermedia MEFM601-06 EU752012 V. intermedia MEFM602-06 EU752013 V. intermedia MEFM603-06 EU752014 V. intermedia MEFM604-06 EU752015 Vieja maculicauda MEFM535-06 EU752016 V. maculicauda MEFM536-06 EU752017 V. maculicauda MEFM537-06 EU752018 V. maculicauda MEFM550-06 EU752019 V. maculicauda MEFM551-06 EU752020 V. maculicauda MEFM552-06 EU752021 V. maculicauda MEFM553-06 EU752022 V. maculicauda MEFM554-06 EU752023 V. maculicauda MEFM555-06 EU752024 V. maculicauda MEFM556-06 EU752025 Vieja synspila MEFM022-05 EU752026 V. synspila MEFM024-05 EU752027 V. synspila MEFM023-05 EU752028 V. synspila MEFM005-05 EU752029 V. synspila MEFM647-06 EU752030 V. synspila MEFM648-06 EU752031 V. synspila MEFM533-06 EU752032 V. synspila MEFM534-06 EU752033 Xiphophorus alvarezi MEFM623-06 EU752034 X. alvarezi MEFM624-06 EU752035 X. alvarezi MEFM625-06 EU752036 Xiphophorus hellerii MEFM609-06 EU752037 X. hellerii MEFM562-06 EU752038 X. hellerii MEFM563-06 EU752039 X. hellerii MEFM564-06 EU752040 X. hellerii MEFM565-06 EU752041 X. hellerii MEFM566-06 EU752042 X. hellerii MEFM605-06 EU752043 X. hellerii MEFM606-06 EU752044 X. hellerii MEFM607-06 EU752045 Xiphophorus maculatus MEFM187-05 EU752046

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 FRESHWATER FISH BARCODES 397

APPENDIX I. Continued

Species BOLD process ID GenBank ID

X. maculatus MEFM188-05 EU752047 X. maculatus MEFM190-05 EU752048 X. maculatus MEFM189-05 EU752049 X. maculatus MEFM191-05 EU752050 X. maculatus MEFM628-06 EU752051 X. maculatus MEFM629-06 EU752052

APPENDIX II. Complete neighbour-joining tree using K2P distances, including 428 COI sequences for 61 freshwater fish species

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 398 M. VALDEZ-MORENO ET AL.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 FRESHWATER FISH BARCODES 399

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 400 M. VALDEZ-MORENO ET AL.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 FRESHWATER FISH BARCODES 401

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402 402 M. VALDEZ-MORENO ET AL.

# 2009 The Authors Journal compilation # 2009 The Fisheries Society of the British Isles, Journal of Fish Biology 2009, 74, 377–402