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Molecular systematics of the New World clingfish genus Gobiesox (Teleostei: Gobiesocidae) and the origin of a freshwater clade
Accepted Manuscript Molecular systematics of the New World clingfish genus Gobiesox (Teleostei: Gobiesocidae) and the origin of a freshwater clade Kevin W. Conway, Daemin Kim, Lukas Rüber, Héctor S. Espinosa Pérez, Philip A. Hastings PII: DOI: Reference:
S1055-7903(16)30361-X http://dx.doi.org/10.1016/j.ympev.2017.04.024 YMPEV 5810
To appear in:
Molecular Phylogenetics and Evolution
Received Date: Revised Date: Accepted Date:
23 November 2016 27 April 2017 27 April 2017
Please cite this article as: Conway, K.W., Kim, D., Rüber, L., Espinosa Pérez, H.S., Hastings, P.A., Molecular systematics of the New World clingfish genus Gobiesox (Teleostei: Gobiesocidae) and the origin of a freshwater clade, Molecular Phylogenetics and Evolution (2017), doi: http://dx.doi.org/10.1016/j.ympev.2017.04.024
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Molecular systematics of the New World clingfish genus Gobiesox (Teleostei: Gobiesocidae) and the origin of a freshwater clade Kevin W. Conwaya*, Daemin Kimb, Lukas Rüberc,d, Héctor S. Espinosa Péreze and Philip A. Hastingsf a
Department of Wildlife and Fisheries Sciences and Biodiversity Research and Teaching
Collections,
Texas
A&M
University,
College
Station,
TX
77843,
USA.
Email:
[email protected] b
Graduate Degree Program, Department of Ecology and Evolutionary Biology, Yale University,
New Haven, CT 06511, USA. c
Naturhistorisches Museum der Burgergemeinde Bern, Bernastrasse 15, 3005 Bern, Switzerland.
d e
Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, 3012 Bern, Switzerland.
Colección Nacional de Peces, Instituto de Biología, Universidad Nacional Autónoma de México.
3er. Circuito Exterior s/n, Cd. Universitaria, 04510 México, D. F., México. f
Marine Biology Research Division, Scripps Institution of Oceanography, University of
California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0244, USA. *corresponding author
Abstract
The phylogenetic relationships between marine and freshwater members of the New World clingfish genus Gobiesox are investigated using both mitochondrial and nuclear sequence data. Phylogenetic hypotheses are derived from Bayesian and Maximum Parsimony analyses of a sixgene concatenated data set (2 mitochondrial and 4 nuclear markers; 4098 bp). Gobiesox is paraphyletic, due to the inclusion of Pherallodiscus, in phylogenetic hypotheses resulting from all analyses and its two included species are reassigned to Gobiesox. Within the expanded genus Gobiesox, the freshwater species (G. cephalus, G. juradoensis, G. mexicanus and G. potamius) represent a monophyletic group that is nested inside of a paraphyletic marine group. Based on the monophyly of the freshwater clingfishes, a habitat transition from marine to freshwater is inferred to have occurred only once in the evolutionary history of the group (potentially in the mid-Miocene). Gobiesox is obtained as part of a larger clade of New World clingfishes, including also members of Acyrtops, Acyrtus, Arcos, Rimicola, Sicyases and Tomicodon equivalent to the subfamily Gobiesocinae. The phylogenetic hypotheses obtained are discussed briefly in relation to the two alternative classifications currently in use simultaneously for the Gobiesocidae. A rediagnosis and list of included species is provided for Gobiesox.
Keywords: Marine; Pacific; Central America; Caribbean; Freshwater; Gobiesocinae
1. Introduction
The family Gobiesocidae contains predominantly small-bodied marine fishes found in the intertidal zones of the Atlantic and Indo-Pacific Oceans (Briggs, 1955). Commonly referred to as clingfishes, members of this family possess a remarkable adhesive disc on their ventral surface (formed by elements of the paired-fin girdles; Guitel, 1888) with which they can attach with tenacity to even irregular and heavily fouled substrates (Wainwright et al., 2014; Ditsche et al., 2014). At present, 168 species of clingfishes are recognized (Eschmeyer and Fong, 2017) and and placed into 49 genera (Conway et al., 2015; Fricke et al., 2017). With 30 valid species, the clingfish genus Gobiesox is by far the largest genus of the Gobiesocidae and is restricted to the New World, including both the Pacific (USA [Alaska] to Chile) and Atlantic (USA [New Jersey] to Brazil) coasts of the Americas (Briggs, 1955) in waters of 0–300 meters in depth (Hastings & Conway, 2017). In addition to representing the largest genus of the Gobiesocidae, Gobiesox is also noteworthy for containing both marine (23 spp.) and freshwater species (7 spp.), the latter representing the only freshwater members of the Gobiesocidae (Briggs, 1955; Briggs & Miller, 1960; Espinosa Pérez & Castro-Aguirre, 1996). The freshwater species of Gobiesox are found only at tropical latitudes and inhabit fast flowing rivers and streams draining into the Pacific Ocean (6 spp.), along the western coast of Central America (Mexico to Panama), northern South America (Colombia, including Isla Gorgona, and Ecuador), and Cocos Island, and the Caribbean Sea (1 sp.), along the eastern coast of Central America (from Honduras to Panama), northern South America (Colombia and Venezuela), and the islands of the Greater and Lesser Antilles (Briggs, 1955; Briggs & Miller, 1960; Bussing, 1998; Ferraris, 2003; Phillip et al., 2013; McMahan et al., 2013; SánchezGonzáles et al., 2010; Mercado-Silva et al., 2016). Compared to their marine congeners, relatively little is known about the biology of freshwater clingfishes. In comparison to marine congeners, the freshwater species of Gobiesox achieve much larger body sizes (>130 mm; Briggs, 1955; Mercado-Silva et al., 2016), and represent some of the largest members of the Gobiesocidae. Though freshwater species of Gobiesox lay eggs in freshwater (K.W. Conway pers. obs.; J.A. Valerio, pers. comm.) it is currently unknown whether they complete their entire life cycle in freshwater or exhibit a marine larval phase (i.e., amphidromy; Myers, 1949; McDowall, 1988, 2007).
Briggs (1955) and Briggs & Miller (1960) considered the freshwater species of Gobiesox to represent “closest relatives” and the most “primitive” members of Gobiesox. These precladistic hypotheses where based on several reductive morphological characteristics shared by the freshwater species, including poorly developed incisors along the lower jaw, short dorsal and anal fins with fewer rays than generally present in marine species, poorly developed fleshy lobes surrounding the mouthparts, and larger body sizes compared to the generally smaller marine species of Gobiesox (Briggs, 1955). Resolution of the phylogenetic relationships of these species within the context of other members of Gobiesox will provide insight into the transition between marine and freshwater habitats in clingfishes, whether exclusively from marine to freshwater, or vice versa, and if this represents a single evolutionary event or multiple transitions. In the current classification of Gobiesocidae (sensu Briggs, 1955), Gobiesox is placed within the subfamily Gobiesocinae with nine other genera of marine clingfishes, including the New World Acyrtops, Acyrtus, Arcos, Derilissus, Pherallodiscus, Rimicola, Sicyases and Tomicodon, and the South African endemic Eckloniaichthys (Briggs, 1955; Conway et al., 2015). Among the gobiesocine taxa, the Mexican Pacific coast endemic genus Pherallodiscus (including two species: P. funebris and P. varius) is considered the closest relative of Gobiesox based on a number of shared morphological features, including the shape of the bony elements supporting the adhesive disc, the shape of the anterior nostril flap, and features of upper jaw dentition (Briggs, 1955: 148). Briggs (1955) erected Pherallodiscus for P. funebris (type species, previously assigned to Gobiesox) and the newly described P. varius based on characteristics of adhesive disc papillae, including the absence of papillae along the midline of disc region A and the absence of paired patches of papillae in disc region C, both of which are present in Gobiesox (see Thomson et al., 1979, Fig. 95). The relationships of New World clingfishes in general, as well as the hypothesis of a sister-group relationship between Gobiesox and Pherallodiscus, have yet to be addressed within a broader phylogenetic framework. Here, we utilize a multi-locus approach to evaluate the phylogenetic relationships of Gobiesox and other New World clingfishes. Our primary aim was to (i) assess, for the first time, the relationships between species of Gobiesox, with emphasis on the phylogenetic relationships of the freshwater taxa, and (ii) test the proposed sister-group relationship between Gobiesox and Pherallodiscus.
2. Materials and Methods
2.1. Taxon Sampling Our sampling includes 13 of the 30 currently recognized species of Gobiesox (subfamily Gobiesocinae), including nine of the 23 marine species (G. adustus, G. barbatulus, G. daedaleus, G. maeandricus, G. nigripinnis, G. pinniger, G. punctulatus, G. rhessodon and G. strumosus) and four of the seven freshwater species (G. cephalus, G. juradonensis, G. potamius and G. mexicanus). There has been considerable confusion concerning the name of the freshwater species of Gobiesox inhabiting the Caribbean Sea drainages of Central and South America and the islands of the Greater and Lesser Antilles, which has most commonly been referred to as G. nudus, following Briggs (1955), or more recently as Arcos nudus (e.g., Mercado-Silva et al., 2016). Following Fernholm & Wheeler (1983), we apply the name Gobiesox cephalus to the freshwater species of Gobiesox inhabiting Caribbean Sea drainages throughout Central/South America and the islands of the Greater and Lesser Antilles and apply the name Arcos nudus to a marine clingfish inhabiting shallow coastal areas throughout the western central Atlantic. In addition to Gobiesox, we also included clingfishes belonging to a number of New and Old World genera, including Alabes (Cheilobranchinae), Chorisochismus (Chorisochisminae), Diademichthys, Discotrema, Lepadichthys (Diademichthinae), Aspasmogaster, Cochleoceps, Parvicrepis (Diplocrepinae), Acyrtops, Acyrtus, Arcos, Pherallodiscus, Rimicola, Sicyases, Tomicodon (Gobiesocinae), Apletodon and Lepadogaster (Lepadogastrinae) to assess the relationships of Gobiesox within the Gobiesocidae. As outgroup taxa, we included three members of Blenniidae (Entomacrodus nigricans, Ophioblennius atlanticus and Salarias fasciatus), one member of Grammatidae (Gramma loreto) and one member of Pseudochromidae (Labracinus cyclophthalmus). Our choice of outgroup taxa is based on the results of recent phylogenetic investigations of the Ovalentaria, the so-called “egg-filament clade”, to which the Gobiesocidae is considered a member (Wainwright et al., 2012; Near et al., 2013; Eytan et al. 2015). Tissues utilized in this study were obtained either via fieldwork (and deposited in museum collections) or directly from museum collections (see Table S1 for a list of voucher specimens).
2.2. DNA Isolation and Sequencing Genomic DNA was extracted from muscle tissue or fin clip using a DNeasy Blood and Tissue Extraction Kit (Qiagen, Inc.) in accordance with the manufacturer’s protocols. DNA sequence data were generated for two mitochondrial loci (12S ribosomal RNA [12S] and cytochrome c oxidase subunit I, [CO1]) and four protein-coding nuclear loci (glycosyltransferase [GLYT]; cardiac muscle myosin heavy chain 6 alpha [MYH6]; SH3 and PX domain containing 3 gene [SH3PX3]; and zic family member 1 [ZIC1]). A segment of the mitochondrial COI and 12S genes was amplified using the primers LCO1490 and HC02198 (Folmer et al., 1994) and L1091 and H1478 (Kocher et al., 1989), respectively. Twenty-five microliter Polymerase Chain Reactions (PCR) for mitochondrial genes contained 1x reaction buffer (pH 8.5), 1.5 mM MgCl2, 0.25 mM of each dNTP, 25 pmol of each primer, 0.05 U⁄µL Taq polymerase, and 2 µL of template. Reaction conditions consisted of an initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 30 sec, 41°C for 1 min for COI/47.6°C for 35 sec for 12S, and 72°C for 1 min 30 sec, followed by a final extension of 72°C for 10 min for COI/1 min for 12S. The nuclear loci were amplified using the nested PCR method and primers described in Li et al. (2007). Amplified PCR products were sequenced using the HighThroughput sequencing facilities at Beckman Coulter Genomics (Danvers, MA, USA).
2.2. Sequence Alignment and Phylogenetic Analyses Sequence alignment was performed with MAFFT version 6.903 (Katoh and Toh, 2010) and checked for accuracy manually. Sequences of the protein-coding genes were translated into amino acids, using MEGA 6.0 (Tamura et al., 2013) to check for frame shifts and premature stop codons. A summary of alignment length and base pair composition for each aligned data set is provided in Table 1. The best-fit sequence substitution model and partitioning scheme for each gene was determined using PartitionFinder V.1.1.1 (Lanfear et al., 2012) based on the Bayesian Information Criterion (BIC) and the ‘models = mrbayes’ setting. The Best-fit substitution models obtained for each gene are listed in Table 1. Phylogenetic relationships were investigated based on individual gene datasets and three concatenated data sets (combined [comprising all six genes]; nDNA [comprising four nuclear genes only]; mtDNA [comprising two mitochondrial genes only]) within a Bayesian framework using MrBayes 3.2.3 (Ronquist et al., 2012) and a Maximum Parsimony (MP) framework using
PAUP* v.4.0b10 (Swofford, 2000). For Bayesian analyses, two independent runs of 10 million generations were performed with four chains, sampling trees every 1,000 generations. Convergence was assessed using potential scale reduction factor (PSRF) values in MrBayes, which were close to 1.0, indicating convergence. The tree samples were used to construct a 50 % majority-rule consensus tree after discarding burn-in. The resulting tree was rooted using Labracinus
cyclophthalmus
and
viewed
using
FigTree
v.1.3.1.
(http://tree.bio.ed.ac.uk/software/figtree). For MP analyses of the individual gene datasets and concatenated datasets we used heuristic searches, utilizing tree-bisection and reconnection (TBR) branch swapping, with starting trees obtained by random stepwise addition (# reps 100). The maximum number of trees saved during each run was allowed to automatically increase by 100, and the MULTREES option was in effect. All characters were equally weighted and unordered. Nodal support was estimated using nonparametic bootstrapping (Felsenstein, 1985) for 1000 pseudoreplicates, utilizing “Fast” stepwise addition. Resulting equally parsimonious cladograms were rooted using Labracinus cyclophthalmus, summarized using a strict consensus method, and viewed using FigTree v.1.3.1.
2.3. Divergence Time Estimation Divergence time estimation was carried out using BEAST v.1.8.2 (Drummond et al., 2012) based on the three concatenated datasets (nDNA+mtDNA, nDNA and mtDNA). We conducted analyses under a model of uncorrelated lognormal distribution rate, unlinking substitution and clock models across partitions, and utilized a Birth-Death speciation prior. No fossils are currently assigned with confidence to the Gobiesocidae and in order to estimate the age of particular clades of gobiesocids we opted to use seven secondary calibrations derived from divergence time estimates obtained by Near et al. (2013) relevant to the family Gobiesocidae and its hypothesized sister taxon the Blennioidei (Hastings & Springer, 2009; Lin & Hastings, 2013). Values for each secondary calibration are provided in Table 2 and the phylogenetic placement of each calibration point is shown in Fig. 2 (nodes A–G). Constraints derived from secondary calibrations were enforced using a log-normal distribution prior. Only five constraints could be applied in the analysis of the mtDNA dataset (see Table 2) due to incongruence between the topology of Near et al. (2013) and the topology obtained from the Bayesian analysis of the mtDNA dataset. For each data set, three independent BEAST runs of 200 million generations
were performed, sampling parameters every 10,000 generations. Tracer v.1.5 (Rambaut and Drummond, 2009) was used to check convergence and stationarity, to determine the number of generations discarded as burn-in, and to confirm that effective sample size (ESS) values were over 200. These three runs were combined with LogCombiner v.1.8.0 (part of the BEAST package). The resulting trees were annotated with TreeAnnotator v.1.8.0 (part of the BEAST package) and the final trees were visualized in FigTree v.1.3.1.
3. Results
3.1. Phylogenetic Relationships Relationships between taxa in the phylogenetic hypotheses resulting from the different analyses (Bayesian or MP) of the combined data set were highly congruent (Fig. 1, S1). We focus here predominantly on the results obtained from the analyses of the combined data set (nDNA+mtDNA), with reference to the results obtained from separate analyses of the nDNA, mtDNA, and individual gene data sets (Figs. S2-S4) where there is conflict. In the topologies resulting from the analyses of the combined data set, all New World taxa of clingfishes are obtained as a monophyletic group. This includes members of the genera Acyrtops, Arcos, Gobiesox, Pherallodiscus, Rimicola, Sicyases, and Tomicodon. This New World clade received strong statistical support in the Bayesian analysis of the combined data set (P.P. = 1.0; Fig. 1) and the nDNA dataset (P.P. =0.99; Fig. S2A) but received low support in the MP analysis of the combined data set (bootstrap support [BS] <50%; Fig. S1) and the nDNA data set (BS =67%; Fig. S2B), was not recovered in any analysis of the mtDNA data set (Fig. S3) and is represented in only 4/12 gene trees (Bayesian and MP analyses of GLYT and SH3PX3 only; Fig. S2, S3). The Old World clingfishes are paraphyletic with respect to the New World taxa, with the South African taxon Chorisochismus dentex representing the sister taxon to the New World clade in the topology resulting from the Bayesian analysis of the combined data set only (Fig. 1). Within the New World clade the Giant Chilean clingfish, Sicyases sanguineus, is placed as the sister taxon to the remaining taxa, which are obtained as a strongly supported monophyletic group regardless of method of analyses (P.P. = 1.0; bootstrap support 95%; Fig. 1, S1). Within the more inclusive New World clade (all New World taxa excluding Sicyases), the genus Arcos is the sister taxon to a clade comprising Gobiesox and Pherallodiscus, and Tomicodon is the sister taxon to a clade
comprising Acyrtops and Rimicola. These relationships received strong statistical support in the Bayesian analysis of the combined data set (P.P. = 1.0; Fig. 1) but received low support in the MP analysis of the same dataset (bootstrap support <50%; Fig. S2). Alternative relationships between the members of this more inclusive New World clade are represented in the topologies resulting from the analyses of the nDNA dataset (Fig. S2). In all topologies resulting from the analyses of the combined data sets, Gobiesox is paraphyletic due to the placement of Pherallodiscus funebris, which is obtained in a strongly supported sister-group relationship with G. daedaleus (Fig. 1, S1-S3). This relationship was obtained also in the majority of the analyses of the individual gene data sets, except for ZIC1 (Fig. S2, 3) and MYH6 (MP only; Fig. S3). Within the Gobiesox/Pherallodiscus clade, the four freshwater species of Gobiesox included in the data set represent a strongly supported monophyletic group, which is present in the topologies resulting from all analyses excluding those of the ZIC1 gene dataset (Fig. S4, S5). Relationships between the four freshwater species of Gobiesox were either unresolved in the topologies resulting from the analyses of the different combined and individual gene data sets. In the topology resulting from the Bayesian analysis of the combined data set G. juradoensis is obtained as the sister taxon to the remainder of the freshwater species; a grouping which received strong statistical support in the Bayesian analysis (P.P=0.99; Fig. 1) but low supported in the MP analysis (BS=62%; Fig. S1). The relationships of Gobiesox and the sister-group relationships of the freshwater clade (when recovered) are largely unresolved and conflicting in the topologies resulting from the analyses of the different data sets, though a small number of sister-group relationships are commonplace among the individual gene trees (Fig. S4-5), including: G. maeandricus+G. rhessodon (11/12 gene trees, excluding MP ZIC1 gene tree), G. adustus+G. pinniger (9/12, excluding Bayesian MYH6 and MP MYH6 and SH3PX3), G. strumosus+G. barbatulus (7/12, excluding Bayesian MYH6 and ZIC1 and MP MYH6, SH3PX3 and ZIC1), G. punctulatus+G. nigripinnis (9/11, excluding Bayesian and MP COI [GLYT sequence for G. nigripinnis not obtained]), and the aforementioned G. daedelus+P. funebris (9/12, excluding Bayesian ZIC1 and MP MYH6 and ZIC1).
3.2. Divergence Time Estimates The topology resulting from the BEAST analysis of the combined data set (Fig. 2) was identical to that resulting from the Bayesian analysis of the same data set. Divergence time estimates
(means and 95% Higher Posterior Density [HPD]) for four cladogenetic events relating to (1) the origin of the New World clingfishes, (2) the origin of the freshwater clade of Gobiesox, (3) the earliest divergence in the freshwater clade of Gobiesox, and (4) the cladogenetic event resulting in G. daedaleus and P. funebris obtained from the BEAST MCMC analysis of the combined data set under a relaxed molecular clock are provided in Fig. 2 and Table 2. These results suggest a mid- to late-Eocene origin for the New World clade of clingfishes (mean 39.3 MYA; 95% HPD 50.8–32.0 MYA) and a mid-Miocene origin for the freshwater clade of Gobiesox (mean 14.3 MYA; 95% HPD 6.8–20.8 MYA). The cladogenetic event resulting in G. daedaleus and P. funebris is estimated to have occurred close to the Miocene-Pliocene transition (mean 5.8 MYA; 95% HPD 8.9-3.1 MYA). Divergence time estimates inferred for these events were either earlier (nDNA; Fig. S6) or significantly later (mtDNA; Fig. S7) when the nuclear or mitochondrial gene sequence data were analyzed separately (Table 2).
4. Discussion
4.1 Phylogenetic relationships and the evolution of freshwater clingfishes Briggs (1955:94) and Briggs & Miller (1960:1) considered the freshwater species of Gobiesox to be “closest relatives” and “more primitive than the marine members of this genus”. Though these ideas were pre-phylogenetic, they could be reinterpreted (using phylogenetic methodology) to mean that either: (1) the freshwater and marine species of Gobiesox are sister groups (i.e. each is monophyletic); or (2) marine species of Gobiesox are derived from ancestors that inhabited freshwater (i.e., freshwater species are paraphyletic with respect to marine species). Using a combination of mitochondrial and nuclear loci, we have presented phylogenetic hypotheses in which the freshwater species of Gobiesox represented in our data set form a monophyletic group that is nested inside of a group of marine taxa, including other species of Gobiesox and Pherallodiscus funebris (Fig. 1, S1). This suggests that the freshwater species of Gobiesox are derived from marine ancestors, as suspected by Hulsey & López-Fernández (2011), and that living in freshwater represents a derived condition for clingfishes. Though our coverage of Gobiesox is not exhaustive (13/30 currently recognized species, including 9/23 marine and 4/7 freshwater), the recovery of a single, well-supported clade comprised exclusively of freshwater
taxa suggests that the transition between marine and freshwaters likely occurred only once in the evolutionary history of the group assuming that the three freshwater species of Gobiesox that we have not sampled, viz. G. fulvus (restricted to Isla del Coco), G. fluviatilis (Pacific streams of central Mexico) and G. juniperoserrai (Baja California, Mexico) also are members of this clade. This finding also suggests that the suite of morphological characters exhibited by the freshwater taxa (including poorly developed incisors along the lower jaw, short dorsal and anal fins, and poorly developed fleshy lobes surrounding the mouthparts) that were considered “primitive” by Briggs (1955) are more likely to represent secondary reductions rather than plesiomorphic traits within Gobiesox. The hypothesized single origin of the freshwater clade within Gobiesox is consistent with recent findings for other groups of New World freshwater fishes of marine origin (“marinederived lineages” of Bloom & Lovejoy, 2017) such as anchovies, drums and pufferfishes (e.g., Bloom and Lovejoy 2012; Lo et al., 2015; Santini et al., 2013), but in contrast to other groups such as New World needlefishes (Lovejoy and Collette, 2001) and silversides (Campanella et al., 2015) that are estimated to have invaded freshwaters multiple times independently. Consistent with most other groups of New World freshwater fishes (with the exception of anchovies and silversides; Bloom and Lovejoy 2012; Campanella et al., 2015), it appears that freshwater clingfishes have not made the transition from freshwater back to marine waters. In the topology resulting from the BEAST analysis of the combined data set, the cladogenetic event that separated the lineage containing the freshwater clingfishes from the closest marine relatives is estimated to have occurred ~13 MYA (95% HPD 16.4–9.8 MYA). The majority of diversification events within the freshwater clade of Gobiesox are estimated to have occurred around the Miocene-Pliocene transition. Though the biogeographic history of Central and northern South America is considered to be well understood, we refrain from speculating about the potential geological drivers of diversification in freshwater clingfishes for the time being until more information on life history is available for this group. In particular, clarifying whether freshwater clingfishes complete their entire lifecycle in freshwater or are amphidromous will be a crucial first step for testing biogeographic hypotheses that may explain their current distribution in both Pacific and Caribbean slope drainages of Central and South America, and the Caribbean. Although the larvae of the freshwater species of Gobiesox are unknown, it is likely that at least some of them retain a marine larval stage given their broad
distribution. For example, G. cephalus is found in drainages of South and Central America (Ferraris, 2003), as well as those on islands throughout the Caribbean basin (Phillip et al., 2013). Similarly, G. juradoensis is found in streams of Colombia as well as Isla Gorgona (Briggs, 1955). It is also difficult to imagine a biogeographic scenario leading to the current distribution of members of this clade in the absence of dispersal through marine waters.
4.2. Phylogenetic relationships of Gobiesox and the New World clingfishes Briggs (1955: 148) considered Pherallodiscus (a genus endemic to the Pacific coast of Mexico) to represent the closest relative (i.e., sister taxon) of Gobiesox, based on a number of morphological similarities. In the phylogenetic hypotheses presented herein, Pherallodiscus (represented by P. funebris) is not placed as the sister taxon of Gobiesox but is instead nested inside of the Gobiesox clade, in a strongly supported sister-group relationship with G. daedaleus (Fig. 1, S2). In this scenario of relationships, Gobiesox is rendered paraphyletic by Pherallodiscus. It is of interest that P. funebris was originally described as a member of Gobiesox by Gilbert (1890) but was later transferred to Pherallodiscus by Briggs (1955), based largely on differences in the arrangement of papillae on the adhesive disc, including the absence of papillae along the midline of disc region A and the absence of paired patches of papillae in disc region C; features invariably present in Gobiesox (Briggs, 1955). Based on the phylogenetic hypotheses presented herein the differences in adhesive disc papillae between Pherallodiscus and Gobiesox are likely due to secondary loss in the lineage containing the former, a process that may have happened within a relatively short timeframe based on the results of the divergence time analysis, which dated the cladogenetic event separating P. funebris from G. daedaleus at ~6 MYA (95% HPD 8.9–3.1) (Fig. 2; Table 2). Based on the phylogenetic hypotheses presented herein we transfer P. funebris back to Gobiesox and place Pherallodiscus in the synonymy of Gobiesox. This nomenclatural change requires amendment to the current generic diagnosis of Gobiesox (sensu Briggs, 1955), which is provided at the end of this paper (see section 5). Briggs (1955) placed Gobiesox within his subfamily Gobiesocinae, together with all other New World genera of clingfishes recognized at that time (viz. Acyrtops, Acyrtus, Arcos, Rimicola, Sicyases and Tomicodon) and the South African Ekloniaichthys. Though our study was not designed to test the monophyly of the Gobiesocinae (sensu Briggs, 1955), in the topologies resulting from the analyses of the combined and nDNA data sets we have obtained a clade
comprised solely of New World taxa that is congruent with the Gobiesocinae of Briggs (1955). These results are in conflict with those of a previous molecular phylogenetic investigation of the Gobiesocidae (Almada et al., 2008) which did not obtain the New World taxa (represented by Gobiesox and Arcos [=Acyrtus lanthanum] in that data set) as a monophyletic group based on the analyses of 12S and 16S sequence data, but are congruent with those of another study (Conway et al., 2014), which obtained a New World clade (represent by Acyrtops, Acyrtus, Arcos, Gobiesox, Rimicola, Sicyases and Tomicodon) based on the analyses of COI sequence data, though without bootstrap support. A New World clade of clingfishes comprising members of Acyrtops, Acyrtus, Gobiesox and Tomicodon also was obtained in another recent multilocus phylogenetic study of clingfishes focused on the relationships of a purportedly asymmetrical species of clingfish from the Indo-Pacific by Fricke et al. (2016). The recovery of a New World clade of clingfishes in recent molecular phylogenetic studies is also congruent with the results of a recent morphology-based study (Conway et al., 2015), which proposed putative morphological synapomorphies (derived from features of oral dentition) in support of a monophyletic group of New World taxa equivalent to the Gobiesocinae of Briggs (1955) minus Eckloniaichthys. The phylogenetic relationships of the New World taxa of clingfishes reported herein, though well supported in terms of branch support statistics (at least in the analyses of the combined dataset), are in conflict with those obtained by Conway et al. (2014). For example, Conway et al. (2014) obtained Tomicodon as the sister taxon of the remaining New World taxa based on the analysis of COI sequence data, whereas Sicyases is placed as the sister taxon of the remaining New World taxa in the topologies resulting from the analyses of the combined and nDNA datasets herein (Fig. 1, S1, S2). Our own analyses of COI sequence data (which included many of the same sequences used by Conway et al., 2014) and the mtDNA data set (comprising sequences of COI+12S) did not obtain a New World clade and the relationships of the New World taxa are unresolved (Fig. S3-S5). Despite these differences, there is some consistency in the placement of Gobiesox between the phylogenetic hypotheses derived from the analyses of the combined data set and that of Conway et al. (2014) in that Arcos+Acyrtus is obtained as the sister taxon of Gobiesox, though with variable statistical support (Fig. 1, S1). It is of interest that all members of Gobiesox (excluding G. funebris and G. varius), Arcos, and two of the three members of Acyrtus (A. artius and A. lanthanum) exhibit paired patches of papillae in disc region C, features unique to these taxa amongst gobiesocids (Briggs, 1955). This feature of adhesive
disc papillae may represent a putative morphological synapomorphy in support of this grouping (with absence in the two species of Gobiesox previously assigned to Pherallodiscus and Acyrtus rubiginosus accounted for by secondary loss). Though support is mounting for a monophyletic group of New World clingfishes (Conway et al., 2014, 2015; Fricke et al., 2016; present study), investigation of additional molecular data sets and morphological character complexes (beyond the oral jaws) are still needed to confirm monophyly of this putative New World clade and of the Gobiesocinae (sensu Briggs, 1955). The inclusion of all proposed members of the Gobiesocinae, including Derilissus (the only putative New World member of the Gobiesocinae missing from molecular phylogenetic investigations conducted to date) and Eckloniaichthys (the only non-New World member of Brigg’s Gobiesocinae), in such an investigation will provide a more thorough test of the monophyly of this interesting subfamily of clingfishes. Although the confidence intervals are large, the estimated time of divergence of this clade based on the analyses of the combined data set (~39 MYA [95% HPD 50.8–32.0]; Table 2, node 1 and Fig. 2) roughly corresponds to that estimated for a New World lineage of blennioid fishes (37.6 MYA; Lin and Hastings, 2013). This time period is coincident with the increasing opening of the Atlantic Ocean and separation of the New World and Old World landmasses, but well before the closing of the Tethys Sea corridor at 12–18 MYA (Adams et al., 1999). If these relationship and divergence estimates are confirmed, they support the conclusion of Bellwood and Wainwright (2003) that the east and west Tethyan fish faunas had diverged well before the terminal Tethyan event.
4.3 Remarks on available classifications for the Gobiesocidae Clingfishes have traditionally been divided into nine subfamilies (Aspasminae, Cheilobranchinae, Chorisochisminae,
Diademicthyinae,
Diplocrepinae,
Gobiesocinae,
Haplocylicinae,
Lepadogastrinae and Trachelochisminae) following Briggs (1955) with subsequent modification by Springer & Fraser (1976). Though widely adopted, this nine subfamily classification has received criticism (Böhlke & Robins, 1970; Craig & Randall, 2009; Almada et al., 2008) and an alternative classification comprised of only two subfamilies has been adopted by some authors (e.g., Van der Lann et al., 2014; Nelson et al., 2016), comprising the Cheilobranchinae (genus Alabes) and the Gobiesocinae (all remaining clingfishes; not equivalent to that of Briggs, 1955). In addition to members of the Gobiesocinae (sensu Briggs, 1955), our dataset includes
representatives of the Cheilobranchinae, Chorisochisminae, Diademicthyinae, Diplocrepinae and Lepadogastrinae, and we take this opportunity to comment briefly on the phylogenetic relationships of non-gobiesocine gobiesocids in the phylogenetic hypotheses obtained herein in relation to the two competing classifications. As discussed above, we have obtained a large monophyletic group of New World taxa equivalent to the Gobiesocinae of Briggs (1955). This New World clade is embedded within a paraphyletic group of Old World taxa, including representatives of the Cheilobranchinae, Chorisochisminae, Diademicthyinae, Diplocrepinae and Lepadogastrinae. Of these five subfamilies, only three are represented by more than one taxon in our analysis, including: Diademichthyinae (represented by Diademichthys, Discotrema and Lepadichthys), Diplocrepinae (Aspasmogaster, Cochleoceps and Parvicrepis), and Lepadogastrinae (Apletodon and Lepadogaster). In the topologies resulting from the analyses of the combined data set (Fig. 1, S1), two of these subfamilies correspond to well-supported monophyletic groups (Diademichthyinae and Lepadogastrinae) but the third (Diplocrepinae) is polyphyletic. Perhaps of greatest interest is the position of the Australian shore eels of the genus Alabes (represented by A. hoesei), which is obtained as part of a monophyletic group including Parvicrepis and Cochleoceps (members of the Diplocrepinae). This arrangement is clearly in conflict with the two-subfamily classification adopted recently by several authors (e.g., Van der Lann et al., 2014; Nelson et al., 2016), because Alabes is not recovered as the sister taxon of the remaining clingfishes (a result also obtained recently by Fricke et al., 2016 using different markers and fewer taxa). These results suggest that neither of the alternative classification schemes used currently for clingfishes accurately reflect the evolutionary relationships of these fishes, as both contain non-monophyletic groups. Until a more complete understanding of the phylogenetic relationships of clingfishes becomes available, preferably derived from a combined morphological and molecular investigation of all currently recognized taxa, we recommend the continued use of the nine-subfamily classification scheme based on Briggs (1955), the preferred classification amongst those familiar and currently working with these charismatic fishes (e.g., Hutchins, 2006; Craig & Randall, 2009; Conway et al., 2015; Craig et al., 2015; Fricke et al., 2016). Adding additional subfamilies to this system to accommodate newly erectly monotypic genera (e.g., Protogobiesocidae for Protogobiesox; Fricke et al., 2016) contributes relatively little to the overall goal of revising the current classification and should be avoided.
5. Gobiesox Lacepède, 1800
5.1. Type species Gobiesox cephalus Lacepède, 1800
5.2. Diagnosis A genus of the Gobiesocidae characterized by the following combination of characters: head broad and dorsoventrally compressed; body moderately dorsoventrally compressed anteriorly, becoming increasingly laterally compressed posteriorly; snout and upper lip separated by a welldeveloped premaxillary grove; upper lip widest at center, narrowest at lateral margins; upper and lower lip, anterolateral margin of snout and anteroventrolateral margin of head with fleshy protrusions of variable number and shape, ranging from low fleshy lobes to narrow finger-like projections; gill-membranes united and free from isthmus; fourth gill arch lacking gill filaments; a well-developed fleshy pad located at base on pectoral fin; adhesive disc single; papillae absent from center of disc region A (G. funebris and G. varius) or present (all remaining species); disc region C without papillae (G. funebris and G. varius) or with paired oval to roughly rectangular patches of papillae (all remaining species); dorsal postcleithra with well-developed spine-like process on medial edge; ventral postcleithra distinctly semi-circular shaped bones, with concave margin anteriorly and convex margin posteriorly; dorsal-fin rays 8-20; anal-fin rays 7-15; pectoral-fin rays 18-27; total caudal-fin rays 18-25 (including 5-6+4-5 principal rays, 4-7 dorsal procurrent rays and 4-7 ventral procurrent rays); total number of vertebrae 25-33.
5.3. Included Species 32 species (listed in alphabetical order), including Gobiesox adustus Jordan & Gilbert, 1882, G. aethus (Briggs, 1951), G. barbatulus Starks, 1913, G. canidens (Briggs, 1951), G. cephalus (freshwater [FW]), G. crassicorpus (Briggs, 1951), G. daedaleus Briggs, 1951, G. eugrammus Briggs, 1955, G. fluviatilis Briggs & Miller, 1960, G. fulvus Meek, 1907 (FW), G. funebris Gilbert, 1890, G. juradoensis Fowler, 1944 (FW), G. juniperoserrai Espinosa Pérez & CastroAguirre, 1996 (FW), G. lanceolatus Hastings & Conway, 2017, G. lucayanus Briggs, 1963, G. maeandricus (Girard, 1858), G. marmoratus Jenyns, 1842, G. marijeanae Briggs, 1960, G.
mexicanus Briggs & Miller, 1960 (FW), G. milleri Briggs, 1955, G. multitentaculus (Briggs, 1951), G. nigripinnis (Peters, 1859), G. papillifer Gilbert, 1890, G. pinniger Gilbert, 1890, G. potamius Briggs, 1955 (FW), G. punctulatus (Poey, 1876), G. rhessodon Smith, 1881, G. schultzi Briggs, 1951, G. stenocephalus Briggs, 1955, G. strumosus Cope, 1870, G. varius (Briggs, 1955), and G. woodsi (Schultz, 1944).
6. Conclusions We have presented the first multi-locus phylogenetic investigation of the New World clingfish genus Gobiesox, the only genus of the Gobiesocidae to contain both marine and freshwater members. The results of all analyses conducted herein suggest that Gobiesox is monophyletic only with the inclusion of Pherallodiscus and the latter is placed in the synonymy of the former. The freshwater members of Gobiesox are obtained as a monophyletic group, supporting the idea of a single habitat transition from marine to freshwater in the evolutionary history of Gobiesox. Gobiesox belongs to a larger clade of New World clingfishes that is equivalent to the subfamily Gobiesocinae (sensu Briggs, 1955 with the exclusion of Eckloniaichthys). The phylogenetic hypotheses presented herein also illustrate problems with the two alternative classifications utilized currently for the Gobiesocidae and additional systematic work is needed to further our understanding of the relationships of the clingfishes.
Acknowledgements We would like to thank M. McGrouther (AMS), M. Sabaj-Perez (ANSP), R. Britz (BMNH), A. Bentley (KU), S. Kullander, Bodil Krajrup (NRM), H.J. Walker (SIO), E. Bermingham, H. Prestridge (TCWC), C. Baldwin, J. Williams (USNM), E. Hilton (VIMS), M. Erdmann, C. Griffiths, R. Chabaria, P. Hundt, D. Bloom, H. H. Tan, and B. Victor for making specimens and/or tissue samples available for this study, T. J. Near for sharing information that enabled us to conduct our divergence time estimation analyses, and D. Bloom and one anonymous reviewer for constructive comments that helped improve this study. KWC also thanks H. Prestridge, D. Phillip, and A. Lugo for help with fieldwork in Trinidad and throughout the Caribbean. This research was funded by NSF (IOS 1256793 to KWC), Texas A&M Agrilife Research (TEX09452 to KWC) and the UCSD Academic Senate (to PAH), and represents publication number #### of the Biodiversity Research and Teaching Collections of Texas A&M University.
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Table 1. Summary of the different data sets used for the phylogenetic analysis, Best Fit substitution models as implemented in PartitionFinder V.1.1.1, and details on MP analyses. Values provided include both outgroup and ingroup taxa. Substitution models used in the combined analyses are the same as those listed for individual genes below.
Number of taxa Alignment Average Empirical base pair composition (%) A T G C Constant sites Variable sites Parsimony informative sites
Best fit substitution model MP # Equally parsimonious cladograms Steps CI RI
Combined
COI
12S
39 4098
39 684
39 394
25.3 20.9 25.6 28.2 2495 1603 1238
23.2 31.6 18.4 26.8 372 312 280
-
3 7394 0.339 0.511
Dataset GLYT
MYH6
SH3PX3
ZIC1
33 846
39 665
38 678
39 831
29.6 21.3 22.8 26.3 175 219 162
25.2 19.1 28.6 27.1 417 429 325
27.1 18.4 27.2 27.3 402 263 203
25.2 18.8 27.1 28.9 465 213 161
23.7 17.3 27.7 31.3 663 168 110
SYM+G/F81+I/H KY+G
SIM+I+G[1st+2nd +3rd]
SYM+G[1st+2nd]/GTR +G[3rd]
K81+I+GTR+G/K8 0+I+G
K80+I+G[1st+2nd]/HKY +G[3rd]
JC/JC+I/HK Y+G
2 2997 0.194 0.317
10 1038 0.346 0.495
1 1292 0.519 0.708
107 897 0.448 0.661
36 601 0.506 0.721
102 289 0.675 0.816
Table 2. Summary of secondary calibrations (mean node height, lower and upper bounds) used in the BEAST analyses of the combined, nDNA and mtDNA data sets and inferred divergence times (median and 95% highest posterior density [HPD]) for three cladogenetic events (nodes 1– 3) in the resulting chronogram. Node letters (A-G) and numbers (1–3) correspond to those in Figures 2, S6, S7. Secondary calibrations are taken from Near et al. (2013). An ‘X’ indicates that a particular constraint could not be enforced.
Node
Calibration
Combined
nDNA
mtDNA
A
80.3(76.9, 83.5)
-
-
-
B
75.8(72.4, 79.5)
-
-
-
C
69.9(66.6, 74.0)
-
-
-
D
37.1(32.8, 41.7)
-
-
-
E
33.3(38.8, 27.6)
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-
X
F
42.9(35.6, 48.7)
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-
-
G
23.1(16.8, 30.0)
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-
X
1
-
39.3(32.0–50.8)
38.6(34.2–45.9)
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2
-
14.3(6.8–20.8)
11.7(8.7–14.9)
43.6(36.0–51.6)
3
-
5.8(3.1–8.9)
4.8(2.4–7.6)
16.7(9.4–24.7)
Figure Captions
Figure 1. Phylogram obtained from Bayesian analysis of the concatenated data set. Numbers above branches represent posterior probabilities obtained from Bayesian analysis of the concatenated data set and bootstrap values obtained from the Maximum Parsimony analysis of the same data set (PP/BS). Marine and freshwater species of Gobiesox are highlighted with a blue and red box, respectively. Photograph of Lepadichthys frenatus by Kenji Sorita (KPMNR0026999).
Figure 2. Chronogram estimated from the Beast analysis of the combined data set utilizing seven calibrations (red bars) derived from Near et al. (2013). Branch lengths are in millions of years. Horizontal bars represent 95% highest posterior density around calibrated (red) and inferred (blue) divergence time estimates between clades. Letters A-G and numbers 1–3 refer to clades listed in Table 2. Alternating epochs are highlighted in grey. Pliocene and Pleistocene are abbreviated to “P+P” in Epoch bar.
Graphical abstract
Highlights
• • • •
First multi-locus molecular phylogenetic investigation of Gobiesox Freshwater species of Gobiesox are monophyletic suggesting a single habitat transition from marine to freshwaters in the evolutionary history of the group Pherallodiscus is placed in the synonymy of Gobiesox Two competing classifications for the Gobiesocidae are both shown to contain nonmonophyletic groups
S1055-7903(16)30361-X http://dx.doi.org/10.1016/j.ympev.2017.04.024 YMPEV 5810
To appear in:
Molecular Phylogenetics and Evolution
Received Date: Revised Date: Accepted Date:
23 November 2016 27 April 2017 27 April 2017
Please cite this article as: Conway, K.W., Kim, D., Rüber, L., Espinosa Pérez, H.S., Hastings, P.A., Molecular systematics of the New World clingfish genus Gobiesox (Teleostei: Gobiesocidae) and the origin of a freshwater clade, Molecular Phylogenetics and Evolution (2017), doi: http://dx.doi.org/10.1016/j.ympev.2017.04.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Molecular systematics of the New World clingfish genus Gobiesox (Teleostei: Gobiesocidae) and the origin of a freshwater clade Kevin W. Conwaya*, Daemin Kimb, Lukas Rüberc,d, Héctor S. Espinosa Péreze and Philip A. Hastingsf a
Department of Wildlife and Fisheries Sciences and Biodiversity Research and Teaching
Collections,
Texas
A&M
University,
College
Station,
TX
77843,
USA.
Email:
[email protected] b
Graduate Degree Program, Department of Ecology and Evolutionary Biology, Yale University,
New Haven, CT 06511, USA. c
Naturhistorisches Museum der Burgergemeinde Bern, Bernastrasse 15, 3005 Bern, Switzerland.
d e
Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, 3012 Bern, Switzerland.
Colección Nacional de Peces, Instituto de Biología, Universidad Nacional Autónoma de México.
3er. Circuito Exterior s/n, Cd. Universitaria, 04510 México, D. F., México. f
Marine Biology Research Division, Scripps Institution of Oceanography, University of
California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0244, USA. *corresponding author
Abstract
The phylogenetic relationships between marine and freshwater members of the New World clingfish genus Gobiesox are investigated using both mitochondrial and nuclear sequence data. Phylogenetic hypotheses are derived from Bayesian and Maximum Parsimony analyses of a sixgene concatenated data set (2 mitochondrial and 4 nuclear markers; 4098 bp). Gobiesox is paraphyletic, due to the inclusion of Pherallodiscus, in phylogenetic hypotheses resulting from all analyses and its two included species are reassigned to Gobiesox. Within the expanded genus Gobiesox, the freshwater species (G. cephalus, G. juradoensis, G. mexicanus and G. potamius) represent a monophyletic group that is nested inside of a paraphyletic marine group. Based on the monophyly of the freshwater clingfishes, a habitat transition from marine to freshwater is inferred to have occurred only once in the evolutionary history of the group (potentially in the mid-Miocene). Gobiesox is obtained as part of a larger clade of New World clingfishes, including also members of Acyrtops, Acyrtus, Arcos, Rimicola, Sicyases and Tomicodon equivalent to the subfamily Gobiesocinae. The phylogenetic hypotheses obtained are discussed briefly in relation to the two alternative classifications currently in use simultaneously for the Gobiesocidae. A rediagnosis and list of included species is provided for Gobiesox.
Keywords: Marine; Pacific; Central America; Caribbean; Freshwater; Gobiesocinae
1. Introduction
The family Gobiesocidae contains predominantly small-bodied marine fishes found in the intertidal zones of the Atlantic and Indo-Pacific Oceans (Briggs, 1955). Commonly referred to as clingfishes, members of this family possess a remarkable adhesive disc on their ventral surface (formed by elements of the paired-fin girdles; Guitel, 1888) with which they can attach with tenacity to even irregular and heavily fouled substrates (Wainwright et al., 2014; Ditsche et al., 2014). At present, 168 species of clingfishes are recognized (Eschmeyer and Fong, 2017) and and placed into 49 genera (Conway et al., 2015; Fricke et al., 2017). With 30 valid species, the clingfish genus Gobiesox is by far the largest genus of the Gobiesocidae and is restricted to the New World, including both the Pacific (USA [Alaska] to Chile) and Atlantic (USA [New Jersey] to Brazil) coasts of the Americas (Briggs, 1955) in waters of 0–300 meters in depth (Hastings & Conway, 2017). In addition to representing the largest genus of the Gobiesocidae, Gobiesox is also noteworthy for containing both marine (23 spp.) and freshwater species (7 spp.), the latter representing the only freshwater members of the Gobiesocidae (Briggs, 1955; Briggs & Miller, 1960; Espinosa Pérez & Castro-Aguirre, 1996). The freshwater species of Gobiesox are found only at tropical latitudes and inhabit fast flowing rivers and streams draining into the Pacific Ocean (6 spp.), along the western coast of Central America (Mexico to Panama), northern South America (Colombia, including Isla Gorgona, and Ecuador), and Cocos Island, and the Caribbean Sea (1 sp.), along the eastern coast of Central America (from Honduras to Panama), northern South America (Colombia and Venezuela), and the islands of the Greater and Lesser Antilles (Briggs, 1955; Briggs & Miller, 1960; Bussing, 1998; Ferraris, 2003; Phillip et al., 2013; McMahan et al., 2013; SánchezGonzáles et al., 2010; Mercado-Silva et al., 2016). Compared to their marine congeners, relatively little is known about the biology of freshwater clingfishes. In comparison to marine congeners, the freshwater species of Gobiesox achieve much larger body sizes (>130 mm; Briggs, 1955; Mercado-Silva et al., 2016), and represent some of the largest members of the Gobiesocidae. Though freshwater species of Gobiesox lay eggs in freshwater (K.W. Conway pers. obs.; J.A. Valerio, pers. comm.) it is currently unknown whether they complete their entire life cycle in freshwater or exhibit a marine larval phase (i.e., amphidromy; Myers, 1949; McDowall, 1988, 2007).
Briggs (1955) and Briggs & Miller (1960) considered the freshwater species of Gobiesox to represent “closest relatives” and the most “primitive” members of Gobiesox. These precladistic hypotheses where based on several reductive morphological characteristics shared by the freshwater species, including poorly developed incisors along the lower jaw, short dorsal and anal fins with fewer rays than generally present in marine species, poorly developed fleshy lobes surrounding the mouthparts, and larger body sizes compared to the generally smaller marine species of Gobiesox (Briggs, 1955). Resolution of the phylogenetic relationships of these species within the context of other members of Gobiesox will provide insight into the transition between marine and freshwater habitats in clingfishes, whether exclusively from marine to freshwater, or vice versa, and if this represents a single evolutionary event or multiple transitions. In the current classification of Gobiesocidae (sensu Briggs, 1955), Gobiesox is placed within the subfamily Gobiesocinae with nine other genera of marine clingfishes, including the New World Acyrtops, Acyrtus, Arcos, Derilissus, Pherallodiscus, Rimicola, Sicyases and Tomicodon, and the South African endemic Eckloniaichthys (Briggs, 1955; Conway et al., 2015). Among the gobiesocine taxa, the Mexican Pacific coast endemic genus Pherallodiscus (including two species: P. funebris and P. varius) is considered the closest relative of Gobiesox based on a number of shared morphological features, including the shape of the bony elements supporting the adhesive disc, the shape of the anterior nostril flap, and features of upper jaw dentition (Briggs, 1955: 148). Briggs (1955) erected Pherallodiscus for P. funebris (type species, previously assigned to Gobiesox) and the newly described P. varius based on characteristics of adhesive disc papillae, including the absence of papillae along the midline of disc region A and the absence of paired patches of papillae in disc region C, both of which are present in Gobiesox (see Thomson et al., 1979, Fig. 95). The relationships of New World clingfishes in general, as well as the hypothesis of a sister-group relationship between Gobiesox and Pherallodiscus, have yet to be addressed within a broader phylogenetic framework. Here, we utilize a multi-locus approach to evaluate the phylogenetic relationships of Gobiesox and other New World clingfishes. Our primary aim was to (i) assess, for the first time, the relationships between species of Gobiesox, with emphasis on the phylogenetic relationships of the freshwater taxa, and (ii) test the proposed sister-group relationship between Gobiesox and Pherallodiscus.
2. Materials and Methods
2.1. Taxon Sampling Our sampling includes 13 of the 30 currently recognized species of Gobiesox (subfamily Gobiesocinae), including nine of the 23 marine species (G. adustus, G. barbatulus, G. daedaleus, G. maeandricus, G. nigripinnis, G. pinniger, G. punctulatus, G. rhessodon and G. strumosus) and four of the seven freshwater species (G. cephalus, G. juradonensis, G. potamius and G. mexicanus). There has been considerable confusion concerning the name of the freshwater species of Gobiesox inhabiting the Caribbean Sea drainages of Central and South America and the islands of the Greater and Lesser Antilles, which has most commonly been referred to as G. nudus, following Briggs (1955), or more recently as Arcos nudus (e.g., Mercado-Silva et al., 2016). Following Fernholm & Wheeler (1983), we apply the name Gobiesox cephalus to the freshwater species of Gobiesox inhabiting Caribbean Sea drainages throughout Central/South America and the islands of the Greater and Lesser Antilles and apply the name Arcos nudus to a marine clingfish inhabiting shallow coastal areas throughout the western central Atlantic. In addition to Gobiesox, we also included clingfishes belonging to a number of New and Old World genera, including Alabes (Cheilobranchinae), Chorisochismus (Chorisochisminae), Diademichthys, Discotrema, Lepadichthys (Diademichthinae), Aspasmogaster, Cochleoceps, Parvicrepis (Diplocrepinae), Acyrtops, Acyrtus, Arcos, Pherallodiscus, Rimicola, Sicyases, Tomicodon (Gobiesocinae), Apletodon and Lepadogaster (Lepadogastrinae) to assess the relationships of Gobiesox within the Gobiesocidae. As outgroup taxa, we included three members of Blenniidae (Entomacrodus nigricans, Ophioblennius atlanticus and Salarias fasciatus), one member of Grammatidae (Gramma loreto) and one member of Pseudochromidae (Labracinus cyclophthalmus). Our choice of outgroup taxa is based on the results of recent phylogenetic investigations of the Ovalentaria, the so-called “egg-filament clade”, to which the Gobiesocidae is considered a member (Wainwright et al., 2012; Near et al., 2013; Eytan et al. 2015). Tissues utilized in this study were obtained either via fieldwork (and deposited in museum collections) or directly from museum collections (see Table S1 for a list of voucher specimens).
2.2. DNA Isolation and Sequencing Genomic DNA was extracted from muscle tissue or fin clip using a DNeasy Blood and Tissue Extraction Kit (Qiagen, Inc.) in accordance with the manufacturer’s protocols. DNA sequence data were generated for two mitochondrial loci (12S ribosomal RNA [12S] and cytochrome c oxidase subunit I, [CO1]) and four protein-coding nuclear loci (glycosyltransferase [GLYT]; cardiac muscle myosin heavy chain 6 alpha [MYH6]; SH3 and PX domain containing 3 gene [SH3PX3]; and zic family member 1 [ZIC1]). A segment of the mitochondrial COI and 12S genes was amplified using the primers LCO1490 and HC02198 (Folmer et al., 1994) and L1091 and H1478 (Kocher et al., 1989), respectively. Twenty-five microliter Polymerase Chain Reactions (PCR) for mitochondrial genes contained 1x reaction buffer (pH 8.5), 1.5 mM MgCl2, 0.25 mM of each dNTP, 25 pmol of each primer, 0.05 U⁄µL Taq polymerase, and 2 µL of template. Reaction conditions consisted of an initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 30 sec, 41°C for 1 min for COI/47.6°C for 35 sec for 12S, and 72°C for 1 min 30 sec, followed by a final extension of 72°C for 10 min for COI/1 min for 12S. The nuclear loci were amplified using the nested PCR method and primers described in Li et al. (2007). Amplified PCR products were sequenced using the HighThroughput sequencing facilities at Beckman Coulter Genomics (Danvers, MA, USA).
2.2. Sequence Alignment and Phylogenetic Analyses Sequence alignment was performed with MAFFT version 6.903 (Katoh and Toh, 2010) and checked for accuracy manually. Sequences of the protein-coding genes were translated into amino acids, using MEGA 6.0 (Tamura et al., 2013) to check for frame shifts and premature stop codons. A summary of alignment length and base pair composition for each aligned data set is provided in Table 1. The best-fit sequence substitution model and partitioning scheme for each gene was determined using PartitionFinder V.1.1.1 (Lanfear et al., 2012) based on the Bayesian Information Criterion (BIC) and the ‘models = mrbayes’ setting. The Best-fit substitution models obtained for each gene are listed in Table 1. Phylogenetic relationships were investigated based on individual gene datasets and three concatenated data sets (combined [comprising all six genes]; nDNA [comprising four nuclear genes only]; mtDNA [comprising two mitochondrial genes only]) within a Bayesian framework using MrBayes 3.2.3 (Ronquist et al., 2012) and a Maximum Parsimony (MP) framework using
PAUP* v.4.0b10 (Swofford, 2000). For Bayesian analyses, two independent runs of 10 million generations were performed with four chains, sampling trees every 1,000 generations. Convergence was assessed using potential scale reduction factor (PSRF) values in MrBayes, which were close to 1.0, indicating convergence. The tree samples were used to construct a 50 % majority-rule consensus tree after discarding burn-in. The resulting tree was rooted using Labracinus
cyclophthalmus
and
viewed
using
FigTree
v.1.3.1.
(http://tree.bio.ed.ac.uk/software/figtree). For MP analyses of the individual gene datasets and concatenated datasets we used heuristic searches, utilizing tree-bisection and reconnection (TBR) branch swapping, with starting trees obtained by random stepwise addition (# reps 100). The maximum number of trees saved during each run was allowed to automatically increase by 100, and the MULTREES option was in effect. All characters were equally weighted and unordered. Nodal support was estimated using nonparametic bootstrapping (Felsenstein, 1985) for 1000 pseudoreplicates, utilizing “Fast” stepwise addition. Resulting equally parsimonious cladograms were rooted using Labracinus cyclophthalmus, summarized using a strict consensus method, and viewed using FigTree v.1.3.1.
2.3. Divergence Time Estimation Divergence time estimation was carried out using BEAST v.1.8.2 (Drummond et al., 2012) based on the three concatenated datasets (nDNA+mtDNA, nDNA and mtDNA). We conducted analyses under a model of uncorrelated lognormal distribution rate, unlinking substitution and clock models across partitions, and utilized a Birth-Death speciation prior. No fossils are currently assigned with confidence to the Gobiesocidae and in order to estimate the age of particular clades of gobiesocids we opted to use seven secondary calibrations derived from divergence time estimates obtained by Near et al. (2013) relevant to the family Gobiesocidae and its hypothesized sister taxon the Blennioidei (Hastings & Springer, 2009; Lin & Hastings, 2013). Values for each secondary calibration are provided in Table 2 and the phylogenetic placement of each calibration point is shown in Fig. 2 (nodes A–G). Constraints derived from secondary calibrations were enforced using a log-normal distribution prior. Only five constraints could be applied in the analysis of the mtDNA dataset (see Table 2) due to incongruence between the topology of Near et al. (2013) and the topology obtained from the Bayesian analysis of the mtDNA dataset. For each data set, three independent BEAST runs of 200 million generations
were performed, sampling parameters every 10,000 generations. Tracer v.1.5 (Rambaut and Drummond, 2009) was used to check convergence and stationarity, to determine the number of generations discarded as burn-in, and to confirm that effective sample size (ESS) values were over 200. These three runs were combined with LogCombiner v.1.8.0 (part of the BEAST package). The resulting trees were annotated with TreeAnnotator v.1.8.0 (part of the BEAST package) and the final trees were visualized in FigTree v.1.3.1.
3. Results
3.1. Phylogenetic Relationships Relationships between taxa in the phylogenetic hypotheses resulting from the different analyses (Bayesian or MP) of the combined data set were highly congruent (Fig. 1, S1). We focus here predominantly on the results obtained from the analyses of the combined data set (nDNA+mtDNA), with reference to the results obtained from separate analyses of the nDNA, mtDNA, and individual gene data sets (Figs. S2-S4) where there is conflict. In the topologies resulting from the analyses of the combined data set, all New World taxa of clingfishes are obtained as a monophyletic group. This includes members of the genera Acyrtops, Arcos, Gobiesox, Pherallodiscus, Rimicola, Sicyases, and Tomicodon. This New World clade received strong statistical support in the Bayesian analysis of the combined data set (P.P. = 1.0; Fig. 1) and the nDNA dataset (P.P. =0.99; Fig. S2A) but received low support in the MP analysis of the combined data set (bootstrap support [BS] <50%; Fig. S1) and the nDNA data set (BS =67%; Fig. S2B), was not recovered in any analysis of the mtDNA data set (Fig. S3) and is represented in only 4/12 gene trees (Bayesian and MP analyses of GLYT and SH3PX3 only; Fig. S2, S3). The Old World clingfishes are paraphyletic with respect to the New World taxa, with the South African taxon Chorisochismus dentex representing the sister taxon to the New World clade in the topology resulting from the Bayesian analysis of the combined data set only (Fig. 1). Within the New World clade the Giant Chilean clingfish, Sicyases sanguineus, is placed as the sister taxon to the remaining taxa, which are obtained as a strongly supported monophyletic group regardless of method of analyses (P.P. = 1.0; bootstrap support 95%; Fig. 1, S1). Within the more inclusive New World clade (all New World taxa excluding Sicyases), the genus Arcos is the sister taxon to a clade comprising Gobiesox and Pherallodiscus, and Tomicodon is the sister taxon to a clade
comprising Acyrtops and Rimicola. These relationships received strong statistical support in the Bayesian analysis of the combined data set (P.P. = 1.0; Fig. 1) but received low support in the MP analysis of the same dataset (bootstrap support <50%; Fig. S2). Alternative relationships between the members of this more inclusive New World clade are represented in the topologies resulting from the analyses of the nDNA dataset (Fig. S2). In all topologies resulting from the analyses of the combined data sets, Gobiesox is paraphyletic due to the placement of Pherallodiscus funebris, which is obtained in a strongly supported sister-group relationship with G. daedaleus (Fig. 1, S1-S3). This relationship was obtained also in the majority of the analyses of the individual gene data sets, except for ZIC1 (Fig. S2, 3) and MYH6 (MP only; Fig. S3). Within the Gobiesox/Pherallodiscus clade, the four freshwater species of Gobiesox included in the data set represent a strongly supported monophyletic group, which is present in the topologies resulting from all analyses excluding those of the ZIC1 gene dataset (Fig. S4, S5). Relationships between the four freshwater species of Gobiesox were either unresolved in the topologies resulting from the analyses of the different combined and individual gene data sets. In the topology resulting from the Bayesian analysis of the combined data set G. juradoensis is obtained as the sister taxon to the remainder of the freshwater species; a grouping which received strong statistical support in the Bayesian analysis (P.P=0.99; Fig. 1) but low supported in the MP analysis (BS=62%; Fig. S1). The relationships of Gobiesox and the sister-group relationships of the freshwater clade (when recovered) are largely unresolved and conflicting in the topologies resulting from the analyses of the different data sets, though a small number of sister-group relationships are commonplace among the individual gene trees (Fig. S4-5), including: G. maeandricus+G. rhessodon (11/12 gene trees, excluding MP ZIC1 gene tree), G. adustus+G. pinniger (9/12, excluding Bayesian MYH6 and MP MYH6 and SH3PX3), G. strumosus+G. barbatulus (7/12, excluding Bayesian MYH6 and ZIC1 and MP MYH6, SH3PX3 and ZIC1), G. punctulatus+G. nigripinnis (9/11, excluding Bayesian and MP COI [GLYT sequence for G. nigripinnis not obtained]), and the aforementioned G. daedelus+P. funebris (9/12, excluding Bayesian ZIC1 and MP MYH6 and ZIC1).
3.2. Divergence Time Estimates The topology resulting from the BEAST analysis of the combined data set (Fig. 2) was identical to that resulting from the Bayesian analysis of the same data set. Divergence time estimates
(means and 95% Higher Posterior Density [HPD]) for four cladogenetic events relating to (1) the origin of the New World clingfishes, (2) the origin of the freshwater clade of Gobiesox, (3) the earliest divergence in the freshwater clade of Gobiesox, and (4) the cladogenetic event resulting in G. daedaleus and P. funebris obtained from the BEAST MCMC analysis of the combined data set under a relaxed molecular clock are provided in Fig. 2 and Table 2. These results suggest a mid- to late-Eocene origin for the New World clade of clingfishes (mean 39.3 MYA; 95% HPD 50.8–32.0 MYA) and a mid-Miocene origin for the freshwater clade of Gobiesox (mean 14.3 MYA; 95% HPD 6.8–20.8 MYA). The cladogenetic event resulting in G. daedaleus and P. funebris is estimated to have occurred close to the Miocene-Pliocene transition (mean 5.8 MYA; 95% HPD 8.9-3.1 MYA). Divergence time estimates inferred for these events were either earlier (nDNA; Fig. S6) or significantly later (mtDNA; Fig. S7) when the nuclear or mitochondrial gene sequence data were analyzed separately (Table 2).
4. Discussion
4.1 Phylogenetic relationships and the evolution of freshwater clingfishes Briggs (1955:94) and Briggs & Miller (1960:1) considered the freshwater species of Gobiesox to be “closest relatives” and “more primitive than the marine members of this genus”. Though these ideas were pre-phylogenetic, they could be reinterpreted (using phylogenetic methodology) to mean that either: (1) the freshwater and marine species of Gobiesox are sister groups (i.e. each is monophyletic); or (2) marine species of Gobiesox are derived from ancestors that inhabited freshwater (i.e., freshwater species are paraphyletic with respect to marine species). Using a combination of mitochondrial and nuclear loci, we have presented phylogenetic hypotheses in which the freshwater species of Gobiesox represented in our data set form a monophyletic group that is nested inside of a group of marine taxa, including other species of Gobiesox and Pherallodiscus funebris (Fig. 1, S1). This suggests that the freshwater species of Gobiesox are derived from marine ancestors, as suspected by Hulsey & López-Fernández (2011), and that living in freshwater represents a derived condition for clingfishes. Though our coverage of Gobiesox is not exhaustive (13/30 currently recognized species, including 9/23 marine and 4/7 freshwater), the recovery of a single, well-supported clade comprised exclusively of freshwater
taxa suggests that the transition between marine and freshwaters likely occurred only once in the evolutionary history of the group assuming that the three freshwater species of Gobiesox that we have not sampled, viz. G. fulvus (restricted to Isla del Coco), G. fluviatilis (Pacific streams of central Mexico) and G. juniperoserrai (Baja California, Mexico) also are members of this clade. This finding also suggests that the suite of morphological characters exhibited by the freshwater taxa (including poorly developed incisors along the lower jaw, short dorsal and anal fins, and poorly developed fleshy lobes surrounding the mouthparts) that were considered “primitive” by Briggs (1955) are more likely to represent secondary reductions rather than plesiomorphic traits within Gobiesox. The hypothesized single origin of the freshwater clade within Gobiesox is consistent with recent findings for other groups of New World freshwater fishes of marine origin (“marinederived lineages” of Bloom & Lovejoy, 2017) such as anchovies, drums and pufferfishes (e.g., Bloom and Lovejoy 2012; Lo et al., 2015; Santini et al., 2013), but in contrast to other groups such as New World needlefishes (Lovejoy and Collette, 2001) and silversides (Campanella et al., 2015) that are estimated to have invaded freshwaters multiple times independently. Consistent with most other groups of New World freshwater fishes (with the exception of anchovies and silversides; Bloom and Lovejoy 2012; Campanella et al., 2015), it appears that freshwater clingfishes have not made the transition from freshwater back to marine waters. In the topology resulting from the BEAST analysis of the combined data set, the cladogenetic event that separated the lineage containing the freshwater clingfishes from the closest marine relatives is estimated to have occurred ~13 MYA (95% HPD 16.4–9.8 MYA). The majority of diversification events within the freshwater clade of Gobiesox are estimated to have occurred around the Miocene-Pliocene transition. Though the biogeographic history of Central and northern South America is considered to be well understood, we refrain from speculating about the potential geological drivers of diversification in freshwater clingfishes for the time being until more information on life history is available for this group. In particular, clarifying whether freshwater clingfishes complete their entire lifecycle in freshwater or are amphidromous will be a crucial first step for testing biogeographic hypotheses that may explain their current distribution in both Pacific and Caribbean slope drainages of Central and South America, and the Caribbean. Although the larvae of the freshwater species of Gobiesox are unknown, it is likely that at least some of them retain a marine larval stage given their broad
distribution. For example, G. cephalus is found in drainages of South and Central America (Ferraris, 2003), as well as those on islands throughout the Caribbean basin (Phillip et al., 2013). Similarly, G. juradoensis is found in streams of Colombia as well as Isla Gorgona (Briggs, 1955). It is also difficult to imagine a biogeographic scenario leading to the current distribution of members of this clade in the absence of dispersal through marine waters.
4.2. Phylogenetic relationships of Gobiesox and the New World clingfishes Briggs (1955: 148) considered Pherallodiscus (a genus endemic to the Pacific coast of Mexico) to represent the closest relative (i.e., sister taxon) of Gobiesox, based on a number of morphological similarities. In the phylogenetic hypotheses presented herein, Pherallodiscus (represented by P. funebris) is not placed as the sister taxon of Gobiesox but is instead nested inside of the Gobiesox clade, in a strongly supported sister-group relationship with G. daedaleus (Fig. 1, S2). In this scenario of relationships, Gobiesox is rendered paraphyletic by Pherallodiscus. It is of interest that P. funebris was originally described as a member of Gobiesox by Gilbert (1890) but was later transferred to Pherallodiscus by Briggs (1955), based largely on differences in the arrangement of papillae on the adhesive disc, including the absence of papillae along the midline of disc region A and the absence of paired patches of papillae in disc region C; features invariably present in Gobiesox (Briggs, 1955). Based on the phylogenetic hypotheses presented herein the differences in adhesive disc papillae between Pherallodiscus and Gobiesox are likely due to secondary loss in the lineage containing the former, a process that may have happened within a relatively short timeframe based on the results of the divergence time analysis, which dated the cladogenetic event separating P. funebris from G. daedaleus at ~6 MYA (95% HPD 8.9–3.1) (Fig. 2; Table 2). Based on the phylogenetic hypotheses presented herein we transfer P. funebris back to Gobiesox and place Pherallodiscus in the synonymy of Gobiesox. This nomenclatural change requires amendment to the current generic diagnosis of Gobiesox (sensu Briggs, 1955), which is provided at the end of this paper (see section 5). Briggs (1955) placed Gobiesox within his subfamily Gobiesocinae, together with all other New World genera of clingfishes recognized at that time (viz. Acyrtops, Acyrtus, Arcos, Rimicola, Sicyases and Tomicodon) and the South African Ekloniaichthys. Though our study was not designed to test the monophyly of the Gobiesocinae (sensu Briggs, 1955), in the topologies resulting from the analyses of the combined and nDNA data sets we have obtained a clade
comprised solely of New World taxa that is congruent with the Gobiesocinae of Briggs (1955). These results are in conflict with those of a previous molecular phylogenetic investigation of the Gobiesocidae (Almada et al., 2008) which did not obtain the New World taxa (represented by Gobiesox and Arcos [=Acyrtus lanthanum] in that data set) as a monophyletic group based on the analyses of 12S and 16S sequence data, but are congruent with those of another study (Conway et al., 2014), which obtained a New World clade (represent by Acyrtops, Acyrtus, Arcos, Gobiesox, Rimicola, Sicyases and Tomicodon) based on the analyses of COI sequence data, though without bootstrap support. A New World clade of clingfishes comprising members of Acyrtops, Acyrtus, Gobiesox and Tomicodon also was obtained in another recent multilocus phylogenetic study of clingfishes focused on the relationships of a purportedly asymmetrical species of clingfish from the Indo-Pacific by Fricke et al. (2016). The recovery of a New World clade of clingfishes in recent molecular phylogenetic studies is also congruent with the results of a recent morphology-based study (Conway et al., 2015), which proposed putative morphological synapomorphies (derived from features of oral dentition) in support of a monophyletic group of New World taxa equivalent to the Gobiesocinae of Briggs (1955) minus Eckloniaichthys. The phylogenetic relationships of the New World taxa of clingfishes reported herein, though well supported in terms of branch support statistics (at least in the analyses of the combined dataset), are in conflict with those obtained by Conway et al. (2014). For example, Conway et al. (2014) obtained Tomicodon as the sister taxon of the remaining New World taxa based on the analysis of COI sequence data, whereas Sicyases is placed as the sister taxon of the remaining New World taxa in the topologies resulting from the analyses of the combined and nDNA datasets herein (Fig. 1, S1, S2). Our own analyses of COI sequence data (which included many of the same sequences used by Conway et al., 2014) and the mtDNA data set (comprising sequences of COI+12S) did not obtain a New World clade and the relationships of the New World taxa are unresolved (Fig. S3-S5). Despite these differences, there is some consistency in the placement of Gobiesox between the phylogenetic hypotheses derived from the analyses of the combined data set and that of Conway et al. (2014) in that Arcos+Acyrtus is obtained as the sister taxon of Gobiesox, though with variable statistical support (Fig. 1, S1). It is of interest that all members of Gobiesox (excluding G. funebris and G. varius), Arcos, and two of the three members of Acyrtus (A. artius and A. lanthanum) exhibit paired patches of papillae in disc region C, features unique to these taxa amongst gobiesocids (Briggs, 1955). This feature of adhesive
disc papillae may represent a putative morphological synapomorphy in support of this grouping (with absence in the two species of Gobiesox previously assigned to Pherallodiscus and Acyrtus rubiginosus accounted for by secondary loss). Though support is mounting for a monophyletic group of New World clingfishes (Conway et al., 2014, 2015; Fricke et al., 2016; present study), investigation of additional molecular data sets and morphological character complexes (beyond the oral jaws) are still needed to confirm monophyly of this putative New World clade and of the Gobiesocinae (sensu Briggs, 1955). The inclusion of all proposed members of the Gobiesocinae, including Derilissus (the only putative New World member of the Gobiesocinae missing from molecular phylogenetic investigations conducted to date) and Eckloniaichthys (the only non-New World member of Brigg’s Gobiesocinae), in such an investigation will provide a more thorough test of the monophyly of this interesting subfamily of clingfishes. Although the confidence intervals are large, the estimated time of divergence of this clade based on the analyses of the combined data set (~39 MYA [95% HPD 50.8–32.0]; Table 2, node 1 and Fig. 2) roughly corresponds to that estimated for a New World lineage of blennioid fishes (37.6 MYA; Lin and Hastings, 2013). This time period is coincident with the increasing opening of the Atlantic Ocean and separation of the New World and Old World landmasses, but well before the closing of the Tethys Sea corridor at 12–18 MYA (Adams et al., 1999). If these relationship and divergence estimates are confirmed, they support the conclusion of Bellwood and Wainwright (2003) that the east and west Tethyan fish faunas had diverged well before the terminal Tethyan event.
4.3 Remarks on available classifications for the Gobiesocidae Clingfishes have traditionally been divided into nine subfamilies (Aspasminae, Cheilobranchinae, Chorisochisminae,
Diademicthyinae,
Diplocrepinae,
Gobiesocinae,
Haplocylicinae,
Lepadogastrinae and Trachelochisminae) following Briggs (1955) with subsequent modification by Springer & Fraser (1976). Though widely adopted, this nine subfamily classification has received criticism (Böhlke & Robins, 1970; Craig & Randall, 2009; Almada et al., 2008) and an alternative classification comprised of only two subfamilies has been adopted by some authors (e.g., Van der Lann et al., 2014; Nelson et al., 2016), comprising the Cheilobranchinae (genus Alabes) and the Gobiesocinae (all remaining clingfishes; not equivalent to that of Briggs, 1955). In addition to members of the Gobiesocinae (sensu Briggs, 1955), our dataset includes
representatives of the Cheilobranchinae, Chorisochisminae, Diademicthyinae, Diplocrepinae and Lepadogastrinae, and we take this opportunity to comment briefly on the phylogenetic relationships of non-gobiesocine gobiesocids in the phylogenetic hypotheses obtained herein in relation to the two competing classifications. As discussed above, we have obtained a large monophyletic group of New World taxa equivalent to the Gobiesocinae of Briggs (1955). This New World clade is embedded within a paraphyletic group of Old World taxa, including representatives of the Cheilobranchinae, Chorisochisminae, Diademicthyinae, Diplocrepinae and Lepadogastrinae. Of these five subfamilies, only three are represented by more than one taxon in our analysis, including: Diademichthyinae (represented by Diademichthys, Discotrema and Lepadichthys), Diplocrepinae (Aspasmogaster, Cochleoceps and Parvicrepis), and Lepadogastrinae (Apletodon and Lepadogaster). In the topologies resulting from the analyses of the combined data set (Fig. 1, S1), two of these subfamilies correspond to well-supported monophyletic groups (Diademichthyinae and Lepadogastrinae) but the third (Diplocrepinae) is polyphyletic. Perhaps of greatest interest is the position of the Australian shore eels of the genus Alabes (represented by A. hoesei), which is obtained as part of a monophyletic group including Parvicrepis and Cochleoceps (members of the Diplocrepinae). This arrangement is clearly in conflict with the two-subfamily classification adopted recently by several authors (e.g., Van der Lann et al., 2014; Nelson et al., 2016), because Alabes is not recovered as the sister taxon of the remaining clingfishes (a result also obtained recently by Fricke et al., 2016 using different markers and fewer taxa). These results suggest that neither of the alternative classification schemes used currently for clingfishes accurately reflect the evolutionary relationships of these fishes, as both contain non-monophyletic groups. Until a more complete understanding of the phylogenetic relationships of clingfishes becomes available, preferably derived from a combined morphological and molecular investigation of all currently recognized taxa, we recommend the continued use of the nine-subfamily classification scheme based on Briggs (1955), the preferred classification amongst those familiar and currently working with these charismatic fishes (e.g., Hutchins, 2006; Craig & Randall, 2009; Conway et al., 2015; Craig et al., 2015; Fricke et al., 2016). Adding additional subfamilies to this system to accommodate newly erectly monotypic genera (e.g., Protogobiesocidae for Protogobiesox; Fricke et al., 2016) contributes relatively little to the overall goal of revising the current classification and should be avoided.
5. Gobiesox Lacepède, 1800
5.1. Type species Gobiesox cephalus Lacepède, 1800
5.2. Diagnosis A genus of the Gobiesocidae characterized by the following combination of characters: head broad and dorsoventrally compressed; body moderately dorsoventrally compressed anteriorly, becoming increasingly laterally compressed posteriorly; snout and upper lip separated by a welldeveloped premaxillary grove; upper lip widest at center, narrowest at lateral margins; upper and lower lip, anterolateral margin of snout and anteroventrolateral margin of head with fleshy protrusions of variable number and shape, ranging from low fleshy lobes to narrow finger-like projections; gill-membranes united and free from isthmus; fourth gill arch lacking gill filaments; a well-developed fleshy pad located at base on pectoral fin; adhesive disc single; papillae absent from center of disc region A (G. funebris and G. varius) or present (all remaining species); disc region C without papillae (G. funebris and G. varius) or with paired oval to roughly rectangular patches of papillae (all remaining species); dorsal postcleithra with well-developed spine-like process on medial edge; ventral postcleithra distinctly semi-circular shaped bones, with concave margin anteriorly and convex margin posteriorly; dorsal-fin rays 8-20; anal-fin rays 7-15; pectoral-fin rays 18-27; total caudal-fin rays 18-25 (including 5-6+4-5 principal rays, 4-7 dorsal procurrent rays and 4-7 ventral procurrent rays); total number of vertebrae 25-33.
5.3. Included Species 32 species (listed in alphabetical order), including Gobiesox adustus Jordan & Gilbert, 1882, G. aethus (Briggs, 1951), G. barbatulus Starks, 1913, G. canidens (Briggs, 1951), G. cephalus (freshwater [FW]), G. crassicorpus (Briggs, 1951), G. daedaleus Briggs, 1951, G. eugrammus Briggs, 1955, G. fluviatilis Briggs & Miller, 1960, G. fulvus Meek, 1907 (FW), G. funebris Gilbert, 1890, G. juradoensis Fowler, 1944 (FW), G. juniperoserrai Espinosa Pérez & CastroAguirre, 1996 (FW), G. lanceolatus Hastings & Conway, 2017, G. lucayanus Briggs, 1963, G. maeandricus (Girard, 1858), G. marmoratus Jenyns, 1842, G. marijeanae Briggs, 1960, G.
mexicanus Briggs & Miller, 1960 (FW), G. milleri Briggs, 1955, G. multitentaculus (Briggs, 1951), G. nigripinnis (Peters, 1859), G. papillifer Gilbert, 1890, G. pinniger Gilbert, 1890, G. potamius Briggs, 1955 (FW), G. punctulatus (Poey, 1876), G. rhessodon Smith, 1881, G. schultzi Briggs, 1951, G. stenocephalus Briggs, 1955, G. strumosus Cope, 1870, G. varius (Briggs, 1955), and G. woodsi (Schultz, 1944).
6. Conclusions We have presented the first multi-locus phylogenetic investigation of the New World clingfish genus Gobiesox, the only genus of the Gobiesocidae to contain both marine and freshwater members. The results of all analyses conducted herein suggest that Gobiesox is monophyletic only with the inclusion of Pherallodiscus and the latter is placed in the synonymy of the former. The freshwater members of Gobiesox are obtained as a monophyletic group, supporting the idea of a single habitat transition from marine to freshwater in the evolutionary history of Gobiesox. Gobiesox belongs to a larger clade of New World clingfishes that is equivalent to the subfamily Gobiesocinae (sensu Briggs, 1955 with the exclusion of Eckloniaichthys). The phylogenetic hypotheses presented herein also illustrate problems with the two alternative classifications utilized currently for the Gobiesocidae and additional systematic work is needed to further our understanding of the relationships of the clingfishes.
Acknowledgements We would like to thank M. McGrouther (AMS), M. Sabaj-Perez (ANSP), R. Britz (BMNH), A. Bentley (KU), S. Kullander, Bodil Krajrup (NRM), H.J. Walker (SIO), E. Bermingham, H. Prestridge (TCWC), C. Baldwin, J. Williams (USNM), E. Hilton (VIMS), M. Erdmann, C. Griffiths, R. Chabaria, P. Hundt, D. Bloom, H. H. Tan, and B. Victor for making specimens and/or tissue samples available for this study, T. J. Near for sharing information that enabled us to conduct our divergence time estimation analyses, and D. Bloom and one anonymous reviewer for constructive comments that helped improve this study. KWC also thanks H. Prestridge, D. Phillip, and A. Lugo for help with fieldwork in Trinidad and throughout the Caribbean. This research was funded by NSF (IOS 1256793 to KWC), Texas A&M Agrilife Research (TEX09452 to KWC) and the UCSD Academic Senate (to PAH), and represents publication number #### of the Biodiversity Research and Teaching Collections of Texas A&M University.
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Table 1. Summary of the different data sets used for the phylogenetic analysis, Best Fit substitution models as implemented in PartitionFinder V.1.1.1, and details on MP analyses. Values provided include both outgroup and ingroup taxa. Substitution models used in the combined analyses are the same as those listed for individual genes below.
Number of taxa Alignment Average Empirical base pair composition (%) A T G C Constant sites Variable sites Parsimony informative sites
Best fit substitution model MP # Equally parsimonious cladograms Steps CI RI
Combined
COI
12S
39 4098
39 684
39 394
25.3 20.9 25.6 28.2 2495 1603 1238
23.2 31.6 18.4 26.8 372 312 280
-
3 7394 0.339 0.511
Dataset GLYT
MYH6
SH3PX3
ZIC1
33 846
39 665
38 678
39 831
29.6 21.3 22.8 26.3 175 219 162
25.2 19.1 28.6 27.1 417 429 325
27.1 18.4 27.2 27.3 402 263 203
25.2 18.8 27.1 28.9 465 213 161
23.7 17.3 27.7 31.3 663 168 110
SYM+G/F81+I/H KY+G
SIM+I+G[1st+2nd +3rd]
SYM+G[1st+2nd]/GTR +G[3rd]
K81+I+GTR+G/K8 0+I+G
K80+I+G[1st+2nd]/HKY +G[3rd]
JC/JC+I/HK Y+G
2 2997 0.194 0.317
10 1038 0.346 0.495
1 1292 0.519 0.708
107 897 0.448 0.661
36 601 0.506 0.721
102 289 0.675 0.816
Table 2. Summary of secondary calibrations (mean node height, lower and upper bounds) used in the BEAST analyses of the combined, nDNA and mtDNA data sets and inferred divergence times (median and 95% highest posterior density [HPD]) for three cladogenetic events (nodes 1– 3) in the resulting chronogram. Node letters (A-G) and numbers (1–3) correspond to those in Figures 2, S6, S7. Secondary calibrations are taken from Near et al. (2013). An ‘X’ indicates that a particular constraint could not be enforced.
Node
Calibration
Combined
nDNA
mtDNA
A
80.3(76.9, 83.5)
-
-
-
B
75.8(72.4, 79.5)
-
-
-
C
69.9(66.6, 74.0)
-
-
-
D
37.1(32.8, 41.7)
-
-
-
E
33.3(38.8, 27.6)
-
-
X
F
42.9(35.6, 48.7)
-
-
-
G
23.1(16.8, 30.0)
-
-
X
1
-
39.3(32.0–50.8)
38.6(34.2–45.9)
-
2
-
14.3(6.8–20.8)
11.7(8.7–14.9)
43.6(36.0–51.6)
3
-
5.8(3.1–8.9)
4.8(2.4–7.6)
16.7(9.4–24.7)
Figure Captions
Figure 1. Phylogram obtained from Bayesian analysis of the concatenated data set. Numbers above branches represent posterior probabilities obtained from Bayesian analysis of the concatenated data set and bootstrap values obtained from the Maximum Parsimony analysis of the same data set (PP/BS). Marine and freshwater species of Gobiesox are highlighted with a blue and red box, respectively. Photograph of Lepadichthys frenatus by Kenji Sorita (KPMNR0026999).
Figure 2. Chronogram estimated from the Beast analysis of the combined data set utilizing seven calibrations (red bars) derived from Near et al. (2013). Branch lengths are in millions of years. Horizontal bars represent 95% highest posterior density around calibrated (red) and inferred (blue) divergence time estimates between clades. Letters A-G and numbers 1–3 refer to clades listed in Table 2. Alternating epochs are highlighted in grey. Pliocene and Pleistocene are abbreviated to “P+P” in Epoch bar.
Graphical abstract
Highlights
• • • •
First multi-locus molecular phylogenetic investigation of Gobiesox Freshwater species of Gobiesox are monophyletic suggesting a single habitat transition from marine to freshwaters in the evolutionary history of the group Pherallodiscus is placed in the synonymy of Gobiesox Two competing classifications for the Gobiesocidae are both shown to contain nonmonophyletic groups