Diversification of the genus Apogon (Lacepède, 1801) (Apogonidae: Perciformes) in the tropical eastern Pacific

Accepted Manuscript Diversification of the genus Apogon (Lacepède, 1801) (Apogonidae: Perciformes) in the tropical eastern Pacific Victor Julio Piñeros, Rosa Gabriela Beltrán-López, Carole C. Baldwin, Enrique Barraza, Eduardo Espinoza, Juan Esteban Martínez, Omar DomínguezDomínguez PII: DOI: Reference:

S1055-7903(18)30144-1 https://doi.org/10.1016/j.ympev.2018.12.010 YMPEV 6366

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

10 March 2018 9 November 2018 8 December 2018

Please cite this article as: Julio Piñeros, V., Gabriela Beltrán-López, R., Baldwin, C.C., Barraza, E., Espinoza, E., Esteban Martínez, J., Domínguez-Domínguez, O., Diversification of the genus Apogon (Lacepède, 1801) (Apogonidae: Perciformes) in the tropical eastern Pacific, Molecular Phylogenetics and Evolution (2018), doi: https://doi.org/10.1016/j.ympev.2018.12.010

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Research Paper

Diversification of the genus Apogon (Lacepède, 1801) (Apogonidae: Perciformes) in the tropical eastern Pacific Victor Julio Piñerosa, *, Rosa Gabriela Beltrán-Lópeza, b, Carole C. Baldwinc, Enrique Barrazad, Eduardo Espinozae, Juan Esteban Martínezf and Omar Domínguez-Domíngueza, g a

Laboratorio de Biología Acuática, Facultad de Biología, Universidad Michoacana de San

Nicolás de Hidalgo, Edificio “R” planta baja, Ciudad Universitaria, Morelia, Michoacán, 58030, México. b

Programa Institucional de Doctorado en Ciencias Biologícas, Facultad de Biología,

Universidad Michoacana de San Nicolás de Hidalgo, Edificio “R” planta baja, Ciudad Universitaria, Morelia, Michoacán, 58030, México. c

Department of Vertebrate Zoology, National Museum of Natural History, Smithsonian

Institution, Washington, DC 20560, USA d

Universidad Francisco Gavidia, Instituto de Ciencia, Tenologia e Inovación, Segundo

Nivel, Calle El Progreso Nº 2748, San Salvador, 1101, El Salvador. e

Dirección del Parque Nacional Galápagos, Puerto Ayora, Isla Santa Cruz, Galápagos,

200350, Ecuador f

Red Multitróficas, Instituto de Ecología A.C., Carretera Antigua a Coatepec # 351, El

Haya, Xalapa, Veracruz, 91070, Mexico. g

Laboratorio Nacional de Análisis y Síntesis Ecológica para la Conservación de Recursos

Genéticos de México, Escuela Nacional de Estudios Superiores, Unidad Morelia, Universidad Nacional Autónoma de México, Apartado Postal 27-3 (Xangari), 58089, Morelia, Michoacán, México.

*Corresponding author: Victor Julio Piñeros. e-mail: [email protected]

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Abstract We examined the role of geographic barriers and historical processes on the diversification of Apogon species within the tropical eastern Pacific (TEP). Mitochondrial and nuclear DNA sequences were used in Bayesian and Maximum likelihood analyses to generate a phylogenetic hypothesis for Apogon species. Bayesian inferences were used to date the cladogenetic events. Analyses with BioGeoBEARS were conducted to reconstruct the biogeographic history and ancestral ranges. The phylogenetic results show a monophyletic clade of TEP Apogon species with A. imberbis from the eastern Atlantic as sister species. The two lineages diverged during the Miocene. Within the TEP clade, two subclades diverged at around 11.1 million years ago (Mya): one clusters the coastal continental species (A. pacificus, A. retrosella and A. dovii), and the second clusters the oceanic island species (A. atradorsatus, A. atricaudus and A. guadalupensis). The estimated diversification times of these subclades were 9.8 and 7.1 Mya, respectively. Within each subclade, species divergences occurred during the Pliocene-Pleistocene epochs. The divergent event between the Atlantic A. imberbis and Apogon TEP clade corresponds to the first closure event of the Central American Seaway. The biogeographic history of Apogon within the TEP appears to be the result of vicariant, dispersal and founder events that occurred during the last 11 million years. The vicariant and dispersal events occurred along the mainland and were associated with the origin of the Central American Gap. The founder events could have allowed the invasion of Apogon to TEP island areas and could have been driven by ancient warming oceanic waters, changes in circulation of marine currents, and the presence of seamounts in ancient marine ridges that allowed the settlement of marine biota. These factors may have allowed Apogon lineages to cross the TEP biogeographic barriers at different times, with subsequent genetic isolation.

Keywords: Biogeographic provinces, cryptic fishes, diversification processes, historical biogeography, reef fishes, tropical eastern Pacific Ocean

Acknowledgements We gratefully acknowledge Rodolfo Perez for help with the dating analysis and historical biogeographic analysis, Margarita Yareli Lopez for help in species identification, Philip A.

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Hastings for the sample of A. guadalupensis, Lee Weigt and D. Ross Robertson for coxI sequences of A. atricaudus. The Comisión Nacional de Areas Naturales Protegidas (CONANP) provided logistic support in the field. Samples were collected by the permits 013/2012 PNG (Ecuador), PPF/DGOPA-035/15 (México), 78-Panamá (Panama), R0562015-OT-CONAGEBIO (Costa Rica) and MARN-AIMA-004-2013 (El Salvador).

Funding sources

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT, Grant No. CB-240875) and Universidad Michoacana de San Nicolás de Hidalgo (CIC2013-2017). VJP was supported by a postdoctoral fellowship (Oficio No. 511-6/17-822) from PRODEP program of SEP of Mexican government.

Declarations of interest none.

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1. Introduction

Vicariance, dispersal and extinction are the main evolutionary processes that explain the current spatial and geographic distribution of biodiversity (Sanmartín, 2012). In the ocean, evolutionary divergence of marine lineages has been associated with historical and contemporary geographical and biological factors. Among the main geographical constraints are both hard (e.g., closures of the Central American Seaway and the Arabian land bridge) and soft (e.g., marine currents and geographical distance) barriers that restrict the dispersal of marine organisms and may affect the divergence of several lineages at different times scales (Cowman and Bellwood, 2013). Biological factors such as a species’ dispersal ability may affect its capacity for crossing barriers and subsequently the intensity of genetic connectivity between populations in different geographic areas. Most marine fishes have pelagic eggs and/or larvae that provide potential for widespread dispersal by local currents and gyres (Mora and Sale, 2002). In some species, pelagic larval duration is correlated with the dispersal distance capacity (Bradbury et al., 2008), but in other species self-recruitment prevails (Jones et al., 1999), suggesting that other traits, such as the dispersal capability of adults and juveniles and broad environmental tolerance, could influence connectivity (Luiz et al., 2012). The high variation in dispersal capacity of marine-fish species and their complex interactions with geographic and environmental factors have rendered understanding and explaining a general pattern of speciation and the genetic structure of their populations a major challenge (Palumbi, 1994; Taylor and Hellberg, 2005). Along the western American coasts, the emergence of the Isthmus of Panama (Lessios, 2008; Bacon et al., 2015; O´Dea et al., 2016), the resulting changes in marine currents (Kameo and Sato, 2000) and geographic distance (Lessios and Robertson, 2006) are factors that help explain the distribution of marine species (Lessios, 2008). This region, known as the tropical eastern Pacific (TEP), is a peripheral marine biogeographic region that is bordered on the west by the Eastern Pacific Barrier, which has isolated it from the Indo-Pacific region for the past 65 million years (Griggs and Hey, 1992). On the east, the American continent acted as a land border from 14 to 3 Mya, when the Panama Isthmus closed (Bacon et al., 2015). North and south of the TEP, cold-water currents (between 16 to

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20 ºC isotherm for the coldest months) limit tropical biodiversity (Briggs and Bowen, 2012). The isolation of the TEP has promoted a large number of endemic fish and invertebrate species (Boschi, 2000; Briggs and Bowen, 2012), many of which may have originated by peripatric speciation (Rocha et al., 2008). The TEP features a mainly rocky shoreline that extends from about Bahia Magdalena on the southern Pacific coast of the Baja Californian Peninsula, through the Gulf of California, to the Gulf of Guayaquil at the border between Ecuador and Peru (Briggs and Bowen, 2012). This rocky shore is interrupted by two sandy and muddy gaps named the Sinaloan Gap (which separates the Gulf of California from southern Mexico) and the Central American Gap (which separates Mexico from Central America through the Tehuantepec Isthmus), with 370 and 1000 km of extension, respectively (Hasting, 2000; Robertson and Cramer, 2009). The TEP also includes five oceanic islands or archipelagoes: the Revillagigedo and Galápagos Archipelagoes, and the Clipperton, Cocos and Malpelo Islands, which are separated by 350 to 1080 km from the mainland (Robertson and Cramer, 2009). The oceanographic characteristics of the TEP include upwelling at several points along the shore, mesoscale currents with cyclonic and anticyclonic gyres, a thermal anomaly along the Mexican coast called the “Eastern Pacific Warm Pool,” and the confluence of sea currents with different temperatures mainly in the island areas (Lavin et al., 2006). The presence of sandy/muddy gaps along the continental shoreline, pelagic gaps (island-mainland distance), and environmental variation influence the distribution and biogeography of several TEP marine shorefishes (Hasting, 2000; Robertson and Cramer, 2009). Recently, a biogeographical analysis of the TEP based on the distribution records of 1261 fish species inhabiting shallow waters helped to delimit its biogeographic provinces (Robertson and Cramer, 2009). That study recognized two mainland provinces (Cortez and Panamic) and one or two oceanic provinces depending on the species’ habitats (e.g., reef, soft bottom, pelagic) and nature of residency (e.g., resident or endemic) (Robertson and Cramer, 2009). When two oceanic provinces were recognized, they comprised (1) Clipperton Island with or without the Revillagigedo Archipelago and (2) Galápagos Archipelago, Cocos Island, Malpelo Island and sometimes Gorgona Island (Robertson and Cramer, 2009). There are exceptions to these general biogeographic patterns, however. For

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example, Hastings (2000) showed that the family Chaenopsidae, which comprises species with cryptic habits, clusters in three mainland provinces (Cortez, Mexican and Panamic) that are separated from one another by shore gaps, and in two island provinces (the Galápagos Archipelago and other islands inside the TEP). Most studies investigating divergence patterns of New World marine shorefishes have focused on exploring the effects of the emergence of the Isthmus of Panama in lineage divergence between Atlantic and TEP basins (Tavera et al., 2012; Thacker, 2017). Only a few studies, focused on crustaceans, have explored the effects on speciation of the geographic barriers inside the TEP (Meyers et al., 2013; Marchant et al., 2015). Here we use phylogenetic and biogeographic analyses to explore the role of the TEP barriers as promoters of genetic isolation within the genus Apogon of the cardinalfish family Apogonidae. The Apogonidae comprise approximately 358 species (Mabuchi et al., 2014), the majority of which are small (less than 100 mm in length), mostly nocturnal and planktivores. Their reproductive biology includes the incubation of fertilized eggs in the male´s mouth, and, although some species lack a pelagic larval phase (Neira, 1991), several species have a pelagic larval phase with a duration ranging from 17 to 45 days (Brothers et al 1983; Raventós and Macpherson, 2001; Fisher et al., 2005). Also, some species exhibit a high self-recruitment rate (Hoffman et al., 2005; Gerlach et al., 2007). Recent phylogenetic analyses of the Apogonidae revealed that a basal branching of the genus Apogon sensu stricto is correlated with geography, with three clades comprising the Indo-Pacific, Atlantic and TEP (Mabuchi et al., 2014; Thacker, 2017). In the TEP, there are six species of Apogon, with geographic distributions reported as follows (Fig. 1B): (1) the most northern species is Apogon guadalupensis, distributed along the Pacific Coast of the Baja California Peninsula and Revillagigedo Islands; (2) A. retrosella and (3) A. pacificus are distributed in the Cortez, Mexican and Panamic Provinces, south to Panama and central Peru, respectively; (4) A. dovii is distributed from the southern portion of the Cortez Province to the Panamic Province and Revillagigedo, Galápagos and Cocos Islands; (5) A. atricaudus is distributed in the Revillagigedo Islands, Clipperton Island and from the southern California coast to central Mexico; and (6) A. atradorsatus is distributed in the Galápagos Archipelago, Malpelo and Cocos islands

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(Robertson and Allen 2015). To date, no study has investigated the diversification of the genus Apogon within the TEP or the role of geographic barriers and other processes related to its diversification. The distribution (Fig. 1B) and biology (i.e. mouth-brooding and high selfrecruitment rates), of Apogon species within the TEP suggest a low dispersal capacity among TEP biogeographic provinces. Accordingly, we predicted that the diversification of this genus in the TEP resulted from the appearance of geographical barriers (i.e., Sinaloa Gap, Central American Gap and Pelagic Gap) at different times. Through extensive sampling of all Apogon species throughout the TEP and by analyzing molecular markers with different rates of molecular evolution—mitochondrial DNA (mtDNA) and nuclear DNA (nDNA)—we investigated the geographical patterns of phylogenetic relationships. We then dated the cladogenetic events and conducted a biogeographic historical analysis to elucidate the influence that geographic barriers and historical processes had on species diversification.

2. Materials and methods

2.1. Collection of Samples

We conducted intensive field collections during 2011-2016 on different expeditions made to the TEP. A total of 248 DNA sequences of Apogon species were analyzed in this study, 63 of which were from samples that we collected from 29 reefs (Table S1 and Fig. 1A). Samples were collected with clove oil and a suction tool, using SCUBA. A fin clip of each captured fish was taken and preserved in absolute ethanol. Voucher specimens were deposited at the Ichthyological Collection of the Universidad Michoacana de San Nicolás de Hidalgo (CPUM). The sample of Apogon guadalupensis was donated by the SCRIPPS Institution of Oceanography (Table S1). Western Atlantic samples from the Smithsonian Institution (Table S1) were collected with the fish anesthetic quinaldine sulfate and dip nets. Muscle tissue from the right flank was sampled from each specimen and preserved in saturated salt-DMSO (dimethyl sulfoxide) buffer (Seutin et al., 1991). Sequences of the three molecular markers used in this study for the eastern Atlantic species Apogon

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imberbis, other genera of the Apogonidae family and the outgroup species Elacatinus evelynae (Table S1), were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/). These species were chosen because sequences were available for at least two of the molecular markers used, helping to ensure a robust phylogenetic analysis. Additionally, we downloaded sequences of the mitochondrial gene Cytochrome C Oxidase I (coxI) for all the species used in this study, which were used only in dating analyses due to the fact that they provided only one haplotype per Apogon species (Table S1).

2.2. Laboratory procedures

Total genomic DNA was extracted using a Phenol-chloroform protocol (Sambrook and Russell, 2001). The mitochondrial cytochrome b (cytb) gene and the nuclear genes, Recombination activating gene 1 (RAG1) and Rhodopsin gene (RHO), were amplified using PCR in ARKTIK Thermal Cycler (Thermo Scientific). Primers LA and HA (Dowling et al., 2002) were used to amplify the cytb; primers Rag-1F and Rag-9R (Quenouille et al., 2004) were used to amplify the RAG1, and primers RH193 and RH1073 (Chen et al., 2003) were used to amplify the RHO. PCR reactions consisted of a total volume of 12.5 µL with 1 µL of 50-100 ng DNA template, 4.25 µL of ultrapure water, 6.25 µL of Dream Taq Green PCR Master Mix 2x (Thermo Scientific), and 0.5 µL of 0.2 µM of each primer. The thermal cycler profiles for cytb consisted of an initial denaturation step at 94 °C for 2 min, followed by 35 cycles of 94 °C for 45 s, 52.4 °C for 60 s and 72 °C for 90 s with a final extension at 72 °C for 10 min. The RAG1 profile included an initial denaturation step at 94 °C for 3 min, followed by 34 cycles of 94 °C for 45 s, 58.8 °C for 50 s and 72 °C for 1:50 min with a final extension at 72 °C for 10 min. The RHO profile included an initial denaturation step at 94 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, 57 °C for 50 s and 72 °C for 1:40 min with a final extension at 72 °C for 10 min. Resulting amplicons were purified with the Exo-sap enzymes (Qiagen, Inc.) and sequenced at Macrogen, Seoul, South Korea.

2.3. Sequence alignment and phylogenetic analysis

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Sequences obtained for each molecular marker were visualized and edited with the software MEGA v7.1 (Tamura et al., 2011). For the RAG1 and RHO sequences, heterozygotic individuals were identified through point mutation, and the alleles were separated using the PHASE algorithm with the software DNAsp v5.0 (Librado and Rozas, 2009). The sequences obtained were aligned with MUSCLE routine implemented using the AliView v1.18 software (Larsson, 2014). Recombination of nDNA genes was analyzed with the phi test in SplitTree4 software (Huson and Brayan, 2006), not showing recombination (RAG1: Phi-test p-value = 0.32; RHO Phi-test p-value = 0.89). Three independent datasets were assembled: mitochondrial (cytb) gene dataset, a combinedmatrix of nDNA genes (RAG1 + RHO) with a final alignment of 1,825 base pairs (bp), and a combined matrix with all markers (cytb + RAG1 + RHO) with a final alignment of 2,555 bp. See Table S2 for additional dataset information. All unique sequences were deposited in GenBank (accession numbers MG957634-MG957988). Model selection based on Akaike information criterion (AIC) and optimal partition settings were performed using PartitionFinder v.1.1.0 software (Lanfear et al., 2012) and recovered the best partition by assigning substitution model for each gene. Analyses were performed independently on each dataset for each molecular marker, on a dataset of the nuclear marker concatenated matrix (RAG1 + RHO), and on a dataset of all the markers of the concatenated matrix (cytb + RAG1 + RHO). To generate the phylogenetic hypotheses using the haplotypes of Apogon species, we performed an analysis of Maximum Likelihood (ML) and Bayesian inference (BI). ML analyses were performed with the software RaxMLGUI v1.5. beta (Stamatakis, 2006; Silvestro and Michalak, 2012) using the Generalized Time Reversible substitution model (GTR) with gamma distribution and invariable sites for the concatenated genes matrix, and a bootstrap analysis of 500 replicates. BI analyses were performed in the software MrBayes v3.1.2 (Huelsenbeck and Ronquist, 2001) setting the priors suggested by PartitionFinder to fit the evolutionary model (Table S2). Four Markov chains were used to sample probability space in two simultaneous but independent runs starting from different random trees. We ran 5 x 107 generations, and the samples were obtained every 1000 generations. To evaluate if BI analyses allowed a good sampling of the posterior probability distribution of all the parameters, the Effective Sample Size (ESS) statistic was evaluated using the Tracer 1.5

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software (Rambaut et al., 2014). ESS values greater than 200 suggest that the MCMC chains got a valid estimate of the parameters. The initial 25% of samples (trees and parameter samples) of each MCMC run were discarded as burn-in, and the remaining trees were summarized as posterior probabilities (Pp), values ≥ 0.90 were considered to represent strong support. Performance of analyses of mtDNA, nDNA and concatenated datasets were assessed using the Bayes Factor (BF). BFs were calculated from the estimated harmonic means of likelihood using the sump command in MrBayes. Performance decisions were made based on the 2ln BF criterion, with BF ≥ 10 considered as strong evidence for rejecting the null hypothesis that the concatenated matrix will best explain the data (Kass and Raftery, 1995).

2.4. Dating analysis

To obtain the divergence times among the Apogon TEP species, we used a concatenated matrix database consisting of two mtDNA markers (coxI and cytb) and two nDNA markers (RAG1and RHO) of 33 species of the Apogonidae (see Table S1). To perform this dating analysis, we used BEAST v1.8.3 (Drummond et al., 2012), ran with an uncorrelated lognormal-relaxed clock model and a birth/death speciation prior. We used the range of mutation rate of cytb in teleosts of 0.0075-0.0125 substitutions/site/million year (Bowen and Grant, 1997) to calibrate the molecular clock. Since the mutation rate is not available for the other genes, they were included in the analysis without calibration information. We ran a search for 5 x 107 generations, while trees were sampled every 500 generations. At the end of the analysis, the effective sample size estimate (ESS) for all parameters exceeded 200, and the appropriate burn-in fraction was determined using Tracer 1.5 (Rambaut et al., 2014). The maximum clade credibility consensus for the post burn-in trees was constructed using the TreeAnnotator 1.7.5 (Drummond et al., 2012) and the representation of the results were visualized in FigTree 1.3.1 (Rambaut, 1999).

2.5. Species geographic distribution

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Initially we delimited the geographic distributions of Apogon TEP species using occurrence records published in the biogeographic databases “Shorefishes of the Tropical Eastern Pacific” (Robertson and Allen, 2015) and “Ocean Biogeographic Information System” (OBIS) (Fig. 1B). We then analyzed the geographic information for TEP Apogon species collected in this study (Fig. 1A) and information obtained from databases of several fish collections, including the Fish Collection of the Museum of Zoology of the University of Costa Rica (UCR) , the Smithsonian Institution's National Museum of Natural History (USNM), the Marine Vertebrate Collection of Scripps Institute of Oceanography (SIO), the Ichthyological Collection of the Universidad Michoacana (CPUM) and the fish collection of the California Academy of Sciences (CAS). To avoid the inclusion of vagrant individuals in species’ assignments to marine biogeographic provinces, the criteria for a species to be included in a province was the existence of >10 captured individuals from that province recorded and/or collected over a period of more than two years. Thus, we obtained consensus geographic distributions of Apogon TEP species (Fig. 1C).

2.6. Historical biogeography

To reconstruct the biogeographic history and ancestral ranges of Apogon in the TEP, we analyzed an ultrametric and dichotomous tree previously obtained in the dating analysis in BEAST using the R package BioGeoBEARS (Matzke, 2013). This package uses the likelihood version of the three most common biogeographical history reconstruction methods: dispersal vicariance analysis (DIVA; Ronquist, 1997) as DIVALIKE method, dispersal extinction cladogenesis analysis (DEC; Ree and Smith, 2008), and Bayesian analysis of biogeography (BayArea; Landis et al., 2013) as BAYAREALIKE method. These traditional models of biogeographic reconstruction assume that the daughter species inherit the distribution or part of the distribution of the ancestor (Matzke, 2013). Also, we used a modified version of each model that includes a founder-event speciation (J parameter, or “jump dispersal” parameter), which allows one daughter species to inherit the distribution of the ancestor and another daughter species to disperse to a novel geographic area outside the ancestor’s range (Matzke, 2013; 2014). These models + J are particularly useful in island systems, where founder-event speciation is expected (Matzke, 2014). We

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compared the fit of the dataset to each of the different models using the Akaike information criterion (AIC) (Matzke, 2013; 2014). We used the consensus geographic distribution of Apogon TEP species obtained by this study (Fig. 1C). Abbreviations used for the marine biogeographic provinces of the TEP (Robertson and Cramer, 2009; Hasting, 2000) are as follows: the Atlantic basin (AT), Cortez Province (CP), Mexican Province (MP), Panamic Province (PP), Galápagos Islands (GI), Malpelo Island (MI), Cocos Island (CI), Clipperton Island (CLI), and Revillagigedo Islands (RI) (Table S3). To test the role of the geographic barriers on Apogon TEP biogeographic history reconstruction, we ran three different dispersal models. In the first model, we did not constrain the directionality or timing of dispersal, and we set the maximum number of ancestral areas to three, as this is the current maximum number of areas inhabited by a single species of Apogon TEP species. Also, we did not enforce constraints on the adjacency of the areas, allowing that any lineages were free to disperse from one area to other area without having to disperse to neighboring areas first. This approach was chosen due to the pelagic larval phase present in many marine fishes, including Apogon species (Brothers et al., 1983; Raventós and Macpherson, 2001; Fisher et al., 2005) and follows analytical protocol implemented in other marine-fish studies (Litsios et al., 2012; Santini et al., 2016; Wainwright et al., 2018). We called it Free Dispersal Model (FDM). In the second and third models, Barrier Model 1 (BM1) and Barrier Model 2 (BM2), we constrained the dispersal probabilities in our analyses to test the role of TEP barriers in two different ways. In BM1, we used two different dispersal probabilities based on the presence of the hard barrier of the Isthmus of Panama, with a low dispersal probability (0.01), and the soft barriers of the Sinaloan, Central American and Pelagic Gaps (Robertson and Cramer, 2009; Hasting, 2000) with a high dispersal rate probability (0.5). In BM2, we used a low dispersal probability for the hard barrier (0.01), and two different dispersal probabilities depending of the geographic area within the TEP in which the dispersion occurred: a) If the dispersal occurred among the continental coastal provinces, where the rocky-reef habitat is nearly continuous except for the continental gaps, we used a dispersal probability of 0.5. b) If the dispersal occurred towards offshore islands where the reef habitat is scarce and discrete in comparison to that of the TEP continental margin, we used a dispersal probability of 0.25.

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We used the AIC to determine which model resulted in the highest likelihood score and thus provided the best explanation of the biogeographic data. The AIC values for each of the biogeographic models were ranked to select the model that yields the smallest values of AIC for inferences. Values higher than 2 of AIC between the models were taken as evidence that the new parameter (e.g. different dispersal probabilities) is important (Anderson, 2008).

3. Results 3.1. Phylogenetic analysis

We obtained a total of 83 sequences consisting of 730 base pairs (bp), which showed 325 variable positions for cytb; 90 sequences consisting of 993 bp showed 295 variable positions for RAG1; and 75 sequences consisting of 832 bp showing 184 variable sites for RHO (Table S1). The phylogenetic hypothesis resulting from analysis of the complete concatenated data set (cytb + RAG1 + RHO) revealed that Apogon imberbis from the Atlantic basin is the sister species of the Apogon TEP clade (Bayesian inference posterior probability, Pp = 99; Maximum Likelihood bootstrap support, Bs = 82; Fig. 2). The six Apogon TEP species (hereafter referred to as the TEP clade) formed a highly supported monophyletic clade (Pp = 99, Bs = 90) clustered in two reciprocally monophyletic sister sub-clades. The first sub-clade, sub-clade I, includes the mainland shore species A. pacificus, A. restrosella and A. dovii (Pp = 98, Bs = 70), and the second one, sub-clade II, includes the oceanic species A. atradorsatus, A. atricaudus, and A. guadalupensis (Pp = 100, Bs = 99), with A. guadalupensis embedded within the A. atricaudus lineage. The BF comparison indicates that the analyses using the mtDNA or nDNA matrices did not provide a better explanation of the data that the concatenated (null hypothesis). The BF values for the concatenated vs mtDNA and vs. nDNA were 1.99 and 1.76, respectively. The nuclear (RAG1 and RHO) analyses produced topologies that are congruent with that of the concatenated tree (data not shown). The mitochondrial (cytb) analyses produced topologies that differ from that of the concatenated tree in several respects. Apogon pacificus was recovered as the sister species to all other TEP species + A. imberbis + A.

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maculatus with little or no support. The remaining TEP clade species were clustered in two groups: a sub-clade that includes the mainland shore species A. retrosella and A. dovii (Pp = 100, Bs = 100) and another sub-clade with a polytomy that includes the oceanic species (A. atradorsatus, A. atricaudus), A. imberbis and A. maculatus (data not show).

3.2. Molecular dating

Results of the molecular-clock analysis indicate that the mean time to the most recent common ancestor (TMRCA) of the TEP clade and the Atlantic species was approximately 13.9 Mya (95% HPD: 8.8-19.6 Mya), placing this divergence in the Miocene period (Fig. 3). The divergence between TEP sub-clade I (continental species) and subclade II (island species) also occurred during the Miocene period, 11.1 Mya (95% HPD: 6.7-16.0 Mya) (Fig. 3). The divergence within sub-clade I, which separated A. pacificus from the A. retrosella + A. dovii group, occurred approximately 9.8 Mya (95% HPD: 5.614.6 Mya) (Fig. 3). Within sub-clade II, the divergence between A. atradorsatus and A. atricaudus occurred approximately. 7.1 Mya (95% HPD: 3.1-11.2 Mya) (Fig. 3). Both events occurred during the late Miocene to Pliocene period. The mean divergence between A. retrosella and A. dovii was estimated at 3.6 Mya (95% HPD: 1.1-6.5 Mya) during the Pliocene-Pleistocene period (Fig. 3).

3.3. Geographic distribution

The revised geographical distributions of TEP Apogon species (Fig. 1C) indicate that they are well defined by geographic barriers. The continental species A. retrosella and A. dovii are geographically separated by the Central American Gap. A few individuals collected very close to the borders of the two biogeographic provinces across the Central American Gap from the primary range of each species were considered vagrant. These individuals were collected in several years that coincided with an ENSO (El Niño Southern Oscillations) event. Apogon pacificus has a wide distribution along the TEP continental coast. Apogon atricaudus is confined to the Revillagigedo and Clipperton islands, and A. atradorsatus to Galápagos, Cocos and Malpelo islands, with a few vagrants (between 1 to

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10 individuals) registered in the Cortez and Panamic Provinces and even between TEP islands.

3.4. Historical biogeography

The historical biogeographic reconstruction results obtained of the three dispersal models (FDM, BM1 and BM2) through BioGeoBEARS showed the DIVALIKE methods with the highest likelihood scores (Table 1, Table S4). Although there were no differences between the DIVALIKE and DIVALIKE+J scores, in the other reconstruction methods the scores improved with the inclusion of founder-event speciation (+J) (Table S4). The Barrier Models were better than FDM in having the smallest AIC scores (Table 1), highlighting the role of biogeographic barriers on Apogon TEP diversification. Although the FDM, BM1, and BM2 models show some similar results (Fig. 4, Fig. S1 and Fig. S2, respectively), here we report and discuss the results of the BM2 model because it resulted in the smallest AIC score among the three models (Table 1). The BM2 model revealed a complex history for the Apogon TEP species, with an ancestral geographic distribution that comprised the Atlantic basin and Cortez and Mexican provinces and five main biogeographic events that occurred during the Miocene and Pliocene epoch. In this historical biogeographic hypothesis, vicariance and dispersal affected divergence within the continental Apogon TEP Sub-clade I, and founder events fostered speciation in the Island Apogon TEP Subclade II (Fig. 4). (1) The first biogeographic event occurred at the basal node as the ancestral area split into two areas in the middle Miocene, 14 Mya. One area comprises the Atlantic basin and the ancestral distribution of A. imberbis, and the second comprises CP+MP, the ancestral area of the Apogon TEP clade. (2) The second biogeographic event occurred within the TEP clade, with a founder event from the CP+MP ancestral area to Galápagos Islands (GI). This split resulted in the establishment of the continental (sub-clade I) and oceanic islands (sub-clade II) areas ~12 MYA. Founder events ~11 MYA by the ancestor of subclade II led to a larger ancestral distribution comprising GI + CI + MI.

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(3) Within sub-clade I, A. pacificus dispersed from the ancestral area CP+MP to the Panamic province (PP) at 10 Mya, resulting in the current distribution of A. pacificus in CP+MO+PP. (4) Also within sub-clade I, A. retrosella and A. dovii diverged, which involved dispersal of the ancestor of both species to an area conformed by CP+MP+PP, with a subsequent vicariant event that split the A. retrosella area (CP+MP) from the A. dovii area (PP) at the beginning of the Pliocene, 4 Mya. (5) Within sub-clade II, a founder event resulted in the ancestor of A. atricaudus invading the oceanic islands of Revillagigedo and Clipperton (RI+CLI), near the end of the Miocene, 7Mya.

4. Discussion

Our phylogeny corroborates phylogenetic hypotheses of previous studies in recognizing a monophyletic TEP Apogon clade and a sister-group relationship between the TEP clade and the Atlantic species (Mabuchi et al., 2014; Thacker, 2017). Our study more specifically suggests, as did that of Mabuchi et al. (2014), that A. imberbis from the tropical eastern Atlantic (TEA) is the sister species of the TEP clade. There are several other examples of close phylogenetic relationships between marine-fish lineages of the TEA and TEP regions, including within the genera Bodianus and Halichoeres (Santini et al., 2016; Wainwright et al., 2018). These results suggest that the invasions of lineages from the TEA into the TEP may be common despite the fact that the regions have been separated from one another by 3500 km of open water since 84 Mya (Floeter et al., 2008). Other possible explanations for a sister-group relationship between TEP Apogon species and A. imberbis include a close relationship between the TEP clade and a now-extinct lineage of tropical western Atlantic (TWA) Apogon that was closely related to A. imberbis. The presence of now-extinct TWA Apogon species in the fossil record from the Miocene period (Aguilera and Rodrigues de Aguilera, 1999) lends some support to this hypothesis. Reconstructing the phylogenetic relationships among fossil and modern species would be valuable in understanding the evolutionary history of New World Apogon.

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Our biogeographic results show a complex history highlighting the role of vicariance, dispersal, long-distance colonization and TEP geographic barriers in the diversification and evolution of the Apogon TEP clade (Fig. 4). Dispersal and subsequent vicariant events appear to explain the distribution of mainland Apogon TEP species, whereas founder events resulted in colonization of TEP islands. Variation in timing of the biogeographic events allowed lineages to cross barriers and to colonize new geographic areas along the TEP with subsequent isolation (Fig. 4). These results are similar to those obtained for other marine fishes, songbirds and island plants (Matzke 2013; Litsios et al., 2014; Matzke; 2014; Moyle et al., 2016, Wainwright et al., 2018). The split of the ancestral area into the TEP Apogon clade and the Atlantic species occurred approximately 13.9 Mya, and could be related to the closure of the Central American Seaway (CAS). This date is similar to that shown by Thacker (2017) for the same historic event in Apogon. The dates are also congruent for the Atlantic and eastern Pacific divergence in fishes of the genera Strongylura and Nicholsina (Banford et al., 2004; Robertson et al., 2006), crustaceans of the genus Alpheus (Knowlton and Weigt, 1998) and bivalve mollusks of the genera Arcopsis and Barbatia (Marko, 2002; Marko and Moran, 2009). The closures of the CAS was a complex event, and the date for the final closure is controversial, ranging from 12.9 to less than 1 Mya ((Duque-Caro, 1990; Bacon et al., 2015; O´Dea et al., 2016; Galvan-Quesada et al., 2016). Paleoceanographic and geological reconstruction studies indicate that there were at least three closure events in the CAS that affected the water masses and microbiota interchanges between the TWA and the TEP basins (Duque-Caro, 1990; Kamikuri et al., 2009). The first event was a partial emergence of the Isthmus of Panama, which occurred approximately 13 Mya, and was associated with global cooling due to the rapid growth of the East Antarctic ice sheet and with the intensification of the California current, which influenced the equatorial Pacific region (Duque-Caro, 1990; Kano and Sato, 2000). The second event was a temporal complete closure of the CAS dated between 10.7-9.4 Mya (Kano and Sato, 2000), tied to a major lowering of the sea level by a high latitude cooling (Kamikuri et al., 2009). Afterwards, a reopening of the CAS occurred between 9.8 to 4.2 Mya that allowed the interchange of water between basins, followed by the final closure of the CAS between 3.7 to 3.4 Mya

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(Kamikuri et al., 2009). The divergence between the Apogon TEP clade and Apogon Atlantic species seems to have occurred in the first closure event.

4.1. Apogon TEP Clade

Within the TEP there are three geographic barriers to gene flow for small cryptic reef fishes: the Central American Gap, the Sinaloan Gap and the Pelagic Gap, which have significant effects on marine biota (Hasting, 2000; Robertson and Cramer, 2009). For the genus Apogon, the founder events possibly allowed the crossing of biogeographic barriers along the TEP in different ways and times. Oceanographic conditions from 13.7 to 9.8 Mya in the TEP were characterized by several thermal changes between cool and warm sea-water conditions, followed by a warming of sea water from 9.8 to 4.2 Mya that generated oceanographic conditions similar to those of the ENSO “El Niño” phenomenon (Kano and Sato, 2000; Kamikuri et al., 2009). The warmer oceanographic conditions could have influenced the founder event from the continental ancestral area across the Pelagic Gap to the Galápagos Islands 11 Mya. Subsequent cooling may have limited dispersal capacity and isolated the island lineages of sub-clade II (Figs. 2-4). The arrival to other TEP islands could be explained by the presence of seamounts on the Cocos ridge and the aseismic Cocos, Malpelo, and Carnegie ridges in the Panama basin, dated at 15 to 10 Mya (Castillo et al., 1988; Werner et al., 1999). These seamounts may have been colonized by marine biota prior to the emergence of the islands. During our field sampling we observed that the geographic distributions of TEP Apogon species are more restricted than was previously known (Robertson and Allen, 2015). For example, although the three continental species (A. retrosella, A. dovii and A. pacificus) have been depicted as being widely geographically distributed within the TEP (Fig. 1B), our data suggest that while A. pacificus is widely distributed along the continental coast, A. retrosella and A dovii largely occur on either side of the Central American Gap (Fig. 1). These observations were corroborated by reviewing several fish collections (see Methods), which revealed patterns of geographic distribution similar to those reported in this study. However, we found reports of a few individuals of all Apogon TEP species occurring outside their primary geographic distributions. We consider such

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observations to be of vagrant individuals because they are based on 1 to 10 individuals collected in no more than two consecutive years. Vagrants are not rare occurrences in the TEP, as distributional shifts have been observed in a great number of TEP fish species during ENSO years (Kong and Bolados, 1987; Lea and Rosenblatt, 2000; Victor et al., 2001; Béarez and Jiménez-Prado, 2003). These dispersal events likely occur due to changes in the direction and velocity of marine currents, and due to higher water temperatures during the ENSO phenomenon (Victor et al., 2001). Notably during the El Niño phenomenon of 1997-98, geographical ranges extended for several TEP fishes, including Apogon pacificus, into the warm-temperate waters of southern California (Lea and Rosenblatt, 2000). The same occurred with marine fishes of the Northern Sea due to rising sea-surface temperatures over recent decades (Perry et al., 2005). Delimitation of species’ geographical distributions also could be compromised by the misidentification of the Apogon TEP species. Our field experience indicates that the Apogon specimens are very delicate and lose coloration and scales very easily, hindering the identification of species within a few hours after capture.

4.2. Sub-clade I: TEP continental Apogon

The ancestral area of the continental sub-clade I (A. pacificus, A. retrosella, A. dovii) was CP+MP, presumably resulting from a vicariant event that separated the Atlantic basin of the Pacific mainland coast. A subsequent dispersal event to the PP could have promoted the expansion of the ancestral lineage of A. pacificus along the TEP continental coast. Because the current distribution of A. pacificus includes both the ancestral area of CP + MP as well as PP, it is unclear what led to the divergence of the ancestor of A. pacificus from the ancestor of A. retrosella and A. dovii. The divergence of these clades could have been a sympatric speciation process, which has been documented in several marine fish genera including Hexagrammos (Crow et al., 2010), Halichoeres (Rocha et al., 2005), Hypoplectrus, Haemulon (Bowen et al., 2013) and Elacatinus (Taylor and Hellberg, 2005). In the other lineage, the ancestor of A. retrosella + A. dovii was distributed in an ancestral area encompassing CP+MP+PP (Fig 4), and the divergence between the lineages probably occurred by a vicariant event that separated the CP+MP (A. retrosella area) from

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PP (A. dovii area). During this event, the ancestor of A. dovii was isolated from A. retrosella around 3.6 Mya in the Pliocene-Pleistocene period (Fig. 3; Fig. 4) by the Central American Gap. The strengthened barrier effect of the Central American Gap likely coincided with the Northern Hemisphere Glaciation (NHG) that occurred 3 to 2 Mya (Raymo, 1994). During this period, the marine currents established their modern-day pattern, and the expansion of the northern ice sheet generated a global climate cooling. The global cooling influenced the strength of the upwelling that occurs in the Gulf of Tehuantepec and resulted in a decrease in sea level (Kamikuri et al., 2009). These conditions promoted the decline of the coral reefs in the TEP region (Glynn and Ault, 2000), ecosystems with which apogonid species are highly associated (Gardiner and Jones, 2005). The effects of the Central American Gap on separation and isolation of marine populations have been dated at 2.4 Mya in the labrisomid blenny, Malacoctenus ebisui (Pedraza-Marrón, 2014), a date very close to the hypothesized divergent event between A. retrosella and A. dovii. For crustaceans of the genus Chalamus, however, the divergence in population attributable to this barrier occurred between 150 to 340 thousand years ago (Meyers et al., 2013; Marchant et al., 2015). The Central American Gap has been linked to the regionalization of fauna inside the TEP, mainly in reef fishes (Hasting, 2000; Robertson and Cramer, 2009).

4.3. Sub-clade II: TEP Islands Apogon

Oceanic island species A. atradorsatus and A. atricaudus presumably diverged ~7.1 Mya due to another founder-event from the southern islands of GI+CI+MI to the more northern Revillagigedo (RI) and Clipperton (CLI) islands (Fig. 4). The dispersal of Apogon to the RI could have occurred by a combination of factors that include, again, the ancient warming oceanic waters (Kamikuri et al., 2009), change of circulation of marine currents caused by the uplifting of the Isthmus of Panama during early and middle Miocene (Kameo and Sato, 2000), and the emergence of the seamounts in the Mathematician Ridge (precursor of Socorro Island), which is dated at 8 to 3.5 Mya (Mammerickx et al., 1988). The biological characteristics of Apogon, such as a short pelagic larval phase (Neira, 1991), the philopatry of juvenile individuals (Atema et al., 2002), the site fidelity and homing

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ability of adult apogonids (Marnane, 2000; Hoffman et al., 2005), and the wide geographic distance between the Galápagos and the Revillagigedo islands (2980 km), likely strengthened the effects of species divergence once isolated in the newly invaded area. Our analysis suggests that A. guadalupensis, the most northern of the TEP Apogon species, may be a population of A. atricaudus rather than a distinct species (Fig. 2; Table S3). However, our data include only one sample of A. guadalupensis, making it difficult to reach a robust conclusion about its evolutionary history. Further genetic and morphological studies of this putative species are needed.

5. Conclusions

This work strongly supports a previously proposed monophyletic clade of tropical eastern Pacific species of the cardinalfish genus Apogon. Our results agree with a previous study that suggests that the eastern Atlantic species, A. imberbis, is the sister species of the TEP clade, with the two lineages diverging in the Miocene period during the first temporal Central American Seaway closure. This work provides a framework to help understand the factors that played a role in the diversification of the Apogon genus in the TEP and possibly that of other TEP reef species. The apogonids of the TEP cluster into two major groups based on geography, one group with shore continental distributions and the other one with oceanic island distributions. Diversification events and biogeographic history appear to be related to vicariance, dispersal and founder events that occurred during the last 11 million years in the TEP. The vicariance and dispersal events occurred along the mainland and were associated with the origin of the Central American Gap. The founder events could have allowed the invasion to TEP island areas, helped by ancient warming oceanic waters, changes in circulation of marine currents and the presence of the seamounts in ancient marine ridges. These factors likely contributed to Apogon lineages crossing the TEP biogeographic barriers at different times, with subsequent isolation.

Appendix A. Supplementary materials Additional supporting information may be found in the online version of this article:

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Appendix A. Supplmentary material Supplementary data associated with this article can be found in the online version.

Figure captions

Figure 1. (A) Spatial distribution of Apogon species sampled in the tropical eastern Pacific (TEP) in this study. The biogeographic provinces within the TEP follow Hastings (2000) and Robertson and Cramer (2009). Short black bars denote the boundaries of the Sinaloa Gap and Central American Gap. Numbers codes correspond to those locations shown in Table S1. Color codes are in accordance with the trees shown in Figs. 2 and 4. The image of A. guadalupensis was taken from Robertson and Allen (2015). (B) Geographic distributions of the six TEP Apogon species based on the previously published biogeography databases “Shorefishes of the Tropical Eastern Pacific” (Robertson and Allen, 2015) and “Ocean Biogeographic Information System” (OBIS). (C) Revised geographical distributions of the six TEP Apogon species based on comparisons between the previously published databases and museum collection records and excluding records identified as vagrants (see Methods section).

Figure 2. Bayesian 50% majority-rule consensus tree of haplotypes of Apogon species included in this study derived from the concatenated dataset (cytb, RAG1 and RHO markers). Numbers on branches of the major clades indicate Bayesian posterior probabilities and maximum likelihood bootstrap (Pp/Bs) values. Color codes are as defined in Fig. 1.

Figure 3. Time-calibrated phylogeny of Apogon species based on the multispecies coalescent analyses of concatenated dataset (cytb, coxI and RAG1). Numbers on the nodes indicate the estimated Time to Most Recent Common Ancestor in millions of years ago (Mya). Numbers in parentheses and the horizontal purple bars represent the 95% Highest Posterior Density intervals for each estimated event.

Figure 4. Biogeographic reconstruction (DIVALIKE + J) derived from Barrier Model 2 BioGeoBEARS analysis of Apogon TEP clade species distribution. The numbers show the main biogeographic events. The geographic areas codes are as follows: AT, Atlantic basin; CP, Cortez

Province; MP, Mexican Province; PP, Panamic Province; GI, Galápagos Islands; MI, Malpelo Island; CI, Cocos Island; CLI, Clipperton Island; RI, Revillagigedo Islands.

Table 1. Comparisons among the biogeographic models analyzed in this study (FDM, Free Dispersal Model; BM1, Barrier Model 1; BM2, Barrier Model 2). In bold is showing the best model chosen. Biogeographic Model

BM2

BM1 FMD

Probabilities of dispersion: pd 3 pd: one for hard barrier; one for continental soft barrier; one for pelagic barrier 2 pd: one for hard barrier; one for soft barrier 1 pd: the same to all geographic areas

Method

J parameter

log Likelihood

AIC

Cumulative AIC

DIVALIKE

yes

-14.78

34.62

0

DIVALIKE

yes

-16.24

37.65

3.03

DIVALIKE

yes

18.17

42.74

8.12

Highlights

Apogon TEP species form a monophyletic clade, and its sister species is A. imberbis from the TEA

The Apogon TEP clade comprises a coastal mainland sub-clade and an oceanic islands sub-clade

The divergence and subsequent diversification of Apogon TEP occurred in the last 13.9My

The TEP barriers have an important role in biogeographic history reconstruction of Apogon TEP clade