Unravelling Morphological, Molecular, and Biogeographical Patterns in the Genus Zapteryx (Guitarﬁsh) from the Mexican Paciﬁc

ICES Journal of Marine Science (2017), 74(6), 1630–1638. doi:10.1093/icesjms/fsx021

Original Article Cutting through the Gordian knot: unravelling morphological, molecular, and biogeographical patterns in the genus Zapteryx (guitarﬁsh) from the Mexican Paciﬁc

Ana Castillo-Paez1, Jonathan Sandoval-Castillo2, David Corro-Espinosa3, Javier Tovar-Avila 4, Marıa-Del-Pilar Blanco-Parra5, Nancy C. Saavedra-Sotelo6, Oscar Sosa-Nishizaki7, Felipe Galvan-Magana~ 8 and Axayacatl Rocha-Olivares1* 1Departamento de Oceanografıa Biologica, CICESE, Laboratorio de Ecologıa Molecular, Carretera Ensenada-Tijuana No. 3918, Ensenada, Baja California 22860, Me´xico 2Molecular Ecology Laboratory, School of Biological Sciences, Flinders University, Adelaide SA 5049, Australia 3Instituto Nacional de Pesca, Calzada Sabalo-Cerritos, S/N, contiguo a estero El Yugo, Programa Tiburon, Centro Regional de Investigaciones Pesqueras de Mazatlan, Mazatlan, Sinaloa 82112, Me´xico 4CRIP Bahıa Banderas, Calle Tortuga 1, La Cruz de Huanacaxtle, INAPESCA, Nayarit 63732, Me´xico 5Universidad de Quintana Roo, Blvd. Bahıa s/n esq. Ignacio Comonfort, Del Bosque, Chetumal, Quintana Roo 77019, Me´xico 6Facultad de Ciencias del Mar, Universidad Autonoma de Sinaloa, Paseo Clussen s/n Col, Los Pinos, Mazatlan, Sinaloa 82000, Me´xico 7Departamento de Oceanografıa Biologica, CICESE, Laboratorio de Ecologıa Pesquera, Carretera Ensenada-Tijuana No. 3918, Ensenada, Baja California 22860, Me´xico 8Instituto Politećnico Nacional, Centro Interdisciplinario de Ciencias Marinas, Apartado Postal 592, La Paz, Baja California Sur, CP 23000, Me´xico *Corresponding author: tel: þ52 646 175-0500; e-mail: [email protected] Castillo-Paez, A., Sandoval-Castillo, J., Corro-Espinosa, D., Tovar-Avila, J., Blanco-Parra, M.-D.-P., Saavedra-Sotelo, N. C., Sosa-Nishizaki, O., Galvan-Magana,~ F., and Rocha-Olivares, A. Cutting through the Gordian knot: unravelling morphological, molecular, and biogeographical patterns in the genus Zapteryx (guitarfish) from the Mexican Pacific. – ICES Journal of Marine Science, 74: 1630–1638. Received 12 October 2016; revised 3 February 2017; accepted 6 February 2017; advance access publication 3 March 2017.

Defining species boundaries is important not only for the appropriate attribution of life history and ecological traits but also for sustainable fishery management and for the conservation of biodiversity. Problems arise from taxonomic uncertainty and incorrect species delineation leading to historical misidentification. This is the case of Pacific guitarfishes in the genus Zapteryx. We use a molecular phylogenetic approach combining mitochondrial and nuclear loci to investigate genetic variation in fish along the Mexican Pacific coast. Our analyses reveal a lack of nuclear and mitochondrial distinction between rays identified morphologically as banded guitarfish Z. exasperata and as southern banded guitarfish Z. xyster, casting doubts on the validity of their current systematics. However, we detected two mitochondrial lineages in accordance with the number of species described for the Pacific: a “northern” lineage corresponding to Z. exasperata and a “southern” lineage possibly attributable to Z. xyster. The poorly understood phenotypic plasticity in coloration and size of the evolutionary lineage of Z. exasperata and its apparently wider than currently thought geographic distribution (at least to Oaxaca) are the major sources of confusion regarding the taxonomic and geographic delineation of these nominal species. In light of our findings, eastern Pacific guitarfishes in the genus Zapteryx require a thorough taxonomic revision using morphological and genetic data to unveil what appears to be a complex pattern of diversification. Keywords: evolutionary lineage, guitarfish, mitochondrial DNA, phenotypic plasticity, RAG1, species delimitation.

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Introduction of both species are poorly defined as the result of taxonomic un- Species boundaries are important in many aspects; the identifica- certainty and historical misidentifications (Casper et al., 2009; tion at the species level is necessary not only for appropriate attri- Bizzarro and Kyne, 2015). Morphological distinction between bution of life history and ecological traits, but also for sustainable Pacific species is based on few diagnostic characters: (1) they have fishery management and conservation of biodiversity. Traditional similar coloration but the most conspicuous is that Z. xyster pos- species’ delineations involve the analysis of morphological char- sesses several round yellowish spots as large as the pupil, each acters, which may overlap and be insufficiently robust and infor- spot ocellated with blackish pigmentation, a distinctive spot on mative, causing improper classifications and taxonomic each side of shoulder; a second on pectoral fine near posterior an- confusion (Mendonça et al., 2011; Arlyza et al., 2013). This prob- gle; a third midway between lateral and median line of back; and lem is particularly important in species with K-selected life his- several fainter spots on the anterior back. (2) Disc slightly longer tory attributes (i.e. slow growth rate, late sexual maturation and than broad in Z. exasperata but broader than long in Z. xyster. (3) low fecundity), for which erroneous species delineations increase Z. xyster has the entire lower surface covered with fine shagreen their vulnerability to overexploitation (Arlyza et al., 2013), as is (Jordan and Evermann, 1896). true for most elasmobranchs. This study sheds light on the problematic identifications and Among chondrichthyans, skates (Family Rajidae) have high the delineations of species in the genus Zapteryx in the Mexican species diversity and morphological conservatism hindering spe- Pacific (MP). We adopt a molecular phylogenetic approach ana- cies delimitation (Ebert and Compagno, 2007). For instance, lysing genetic variation at mitochondrial and nuclear loci of fish Himantura uarnak is a species complex, and members of the collected along the MP coast. Our results reveal a lack of genetic group have often been confused because of their overall similarity distinction between rays identified as Z. exasperata and Z. xyster among taxa in the disc shape and dorsal coloration pattern in this geographic region, as well as the presence of a divergent (Arlyza et al., 2013). In this case, morphological diagnostic char- species-specific lineage in the southernmost samples from acters have not been found or are not useful, leading to the com- Oaxaca. The presence of an organism carrying Z. exasperata mon outcome of lumping two or more species together. mtDNA in Oaxaca suggests that both lineages overlap geographi- Molecular markers provide alternative powerful tools to corrobo- cally. Overall, our genetic findings cast doubt on the systematic rate the boundaries of described species and to estimate evolu- validity of the morphological characters used to distinguish Z. tionary relationships among populations (Ovenden et al., 2010). exasperata and Z. xyster, which appear to be plastic among organ- The adoption of molecular methods in elasmobranchs helps to isms sharing northern haplotypes (i.e. predominantly identified clarify aspects such as systematic and phylogenetic relationships, as Z. exasperata). However, our data also support the existence of as well as biogeographical patterns (Dudgeon et al., 2012). For in- genetic divergence between southern organisms, most likely in stance, two species of western Atlantic sharpnose sharks the Z. xyster lineage. A thorough revision of morphological and genetic variation of the genus Zapteryx in the eastern Pacific is re- (Rhizoprionodon terraenovae and R. porosus) show few morpho- quired to thoroughly understand its complex pattern of logical diagnostic characters; being differentiated by an overlap- diversification. ping number of vertebrae. Consequently, their taxonomic status remained uncertain. The level of interspecific mitochondrial Material and methods DNA (mtDNA) divergence was higher than the intraspecific and Sampling reciprocal monophyly of the mtDNA lineages reflected their genetic independence and distinction as different species Fifty-nine muscle or liver samples of Zapteryx spp. specimens were collected from 14 localities along the MP (Table 1). Sixteen (Mendonça et al., 2011). On the other hand, the magpie fiddler specimens were sampled along the Pacific Coast of Baja ray Trygonorrhina melaleuca and the southern fiddler ray T. California (PCBC), 37 in the Gulf of California (GC), and six in dumerilii were described as different species but mtDNA and nu- Oaxaca (Table 1). Samples were preserved in non-denatured clear DNA (nDNA) sequences revealed that T. melaleuca is a rare ethanol (95%) at room temperature until processed in the morph of T. dumerilii possessing a distinctive colour pattern. laboratory. This is of consequence to the management and conservation of these organisms, since T. melaleuca has been listed as an endan- gered species on the IUCN Red List, when in fact, it seems not to Genomic DNA extraction be a valid species but a synonym of T. dumerilli (Donnellan et al., Total genomic DNA was extracted from ca. 100 mg of finely 2015). chopped liver or muscle tissue using either proteinase K digestion Three species of guitarfish have been described in the genus followed by a salting-out protocol with lithium chloride Zapteryx. The shortnose guitarfish, Zapteryx brevirostris Mu¨ller (Gemmell and Akiyama, 1996) or PureLink Genomic DNA Mini and Henle, 1841, is restricted to the Southwestern Atlantic, from Kit (Invitrogen, Life Technologies), particularly for liver tissue, Southern Brazil to the coast of Argentina and Uruguay Wosnick according to the manufacturer protocol. Quality of extraction and Freire, 2013); the banded guitarfish, Z. exasperata Jordan and products was checked with agarose (1.5%) gel electrophoresis Gilbert, 1880, is found from Southern California to Peru (Allen and quantity was estimated spectrophotometrically with a and Robertson, 1994; McEachran and Notarbartolo-Di-Sciara, Nanodrop (Thermo Scientific). DNA was stored in 1X TE buffer 1995); and the southern banded guitarfish, Z. xyster Jordan and pH 8.0 at 20 C and used for the Polymerase Chain Reaction Evermann, 1896, whose distribution extends from Mazatlan, (PCR). Mexico, to Peru (Robertson and Allen, 2002). Ebert (2003) ques- tioned whether Z. exasperata occurred south of Mazatlan, based Amplification and sequencing

on the morphological identiﬁcation of organisms from these lati- Three mitochondrial (NADH dehydrogenase subunit 2 [ND2], tudes as Z. xyster. Present day consensus is that the distributions non-coding duplication remnant region [NCR], and the control-

Table 1. Sampling location of guitarfishes Zapteryx spp. Region Locations N W n Year of collection Morphological identification PCBC 1 Puerto San Carlos 29630 115480 2 2011 Z. exasperata Punta Canoas 29420 115120 4 2011 Z. exasperata 2 Punta Santa Marıa 28940 114510 1 2011 Z. exasperata Laguna Manuela 28420 114080 3 2011 Z. exasperata 3 San Ignacio 26380 113180 3 2011 Z. exasperata Bahıa Magdalena 24290 112360 3 2015 Z. exasperata GC 4 San Felipe 31010 114830 4 2002 Z. exasperata Bahıa los Angeles 28950 113560 1 2002 Z. exasperata 5 Mulege´26530 111580 3 2015 Z. exasperata Bahıa de la Paz 24150 110310 3 2002 Z. exasperata 6 Bahıa Kino 28820 111940 2 2004 Z. exasperata Estero del Soldado 27950 110960 3 2004 Z. exasperata 7 Sinaloa 25790 109350 21 2015 Z. xyster 23580 106740 CP 8 Oaxaca 16530 96240 5 2002 Zapteryx sp. 1 2013 Z. xyster* N, latitude; W, longitude; n, number of individuals collected; PCBC, Pacific Coast of Baja California; GC, Gulf of California; CP, Central Pacific. *Degraded DNA sample.

region [CR]) and one nuclear (RAG1) genes were analysed. The the GTR-C as substitution model. FigTree 1.4.2 (Rambaut and non-coding duplication remnant region was identified from se- Drummond, 2007) was used to draw phylogenies. quencing the entire mitochondrial genome of Z. exasperata (Castillo-Paez et al., 2016). PCRs were carried out in 12.5 ml reac- Results tion mixture with the following concentrations 200 mMof Morphological identification dNTPs, 1 PCR buffer (100 mM Tris HCl pH 8.3, 500 mM KCl, Three morphotypes were detected in the study area (Table 1 and l 15 mM MgCl2, 0.01% gelatine), 0.5 M of each primer Figure 1). Five samples from Oaxaca did not correspond with (Supplementary Table S1), 0.5 U Taq DNA polymerase morphological descriptions for any Zapteryx spp. While these (BioLabs), 0.3 mg/ml BSA (BioLabs), and 20 ng DNA template. specimens showed a longer than wide disk, characteristic of Z. Thermal cycling consisted of: 10 min at 95 C, followed by 35 cy- exasperata (Jordan and Evermann, 1896), they also presented six cles of 30 s at 94C, 30 s at annealing temperature specific to each yellow and blackish ocelli on the dorsal side of the disk, coupled primer pair at (ND2 at 60 C; CR at 62 C; NCR at 53 C RAG1 at with a prominent snout pointed at the tip, characteristics of Z. 68 C) and 60 s at 72 C; and a final extension step of 20 min at xyster (Jordan and Evermann, 1896). 72 C. PCR products were verified in agarose (1.5%) gel electrophoresis and purified using either the QIAquick PCR Purification Kit (Qiagen) or ExoSAP-IT for PCR clean-up (Affymetrix) ac- Patterns of genetic variation cording to manufacturer’s protocol before being cycle sequenced The length of multiple alignments was 984 bp for ND2, 605 bp for using Big Dye Terminator 3.1 chemistry in an ABI 3730xl DNA the CR, 624 bp for the full NCR, and 552 bp for the RAG1gene. sequencer (Applied Biosystems). The NCR showed three or four tandem repeat motifs (100 bp in length), so individual haplotypes varied between 520 and 622 bp in length. NCR was the most polymorphic mitochondrial gene Sequence analyses presenting a large number of variable and parsimony-informative End-trimming and base-calling were conducted using sites and haplotypes. CR was the most conserved gene presenting CodonCode Aligner 3.7.1, (Codon Code Corporation, Dedham, only a few mutations defining five haplotypes. Concatenated MA, USA). Haplotypes were identified considering indels (in- mtDNA sequences produced 33 haplotypes among 58 individuals cluding those from tandem repeat motifs) as a fifth character us- (Table 2); 16 of which were singletons. Some RAG1 sequences ing DnaSP 5 (Librado and Rozas, 2009). Sequence alignment was possessed up to four heterozygous positions of a total of eight carried out with MUSCLE based on UPGMA clustering as imple- variable sites, which defined nine alleles (Table 2). mented in MEGA 6.06 (Tamura et al., 2013) and verified manu- ally. Genetic divergence was computed using PAUP* v.4 Evidence of phenotypic plasticity (Swofford, 2002) with the best-fit model of molecular evolution All individuals from Sinaloa were morphologically identified as Z. estimated in jModelTest 2.1.7 (Darriba et al., 2012). xyster (Table 1 and Figure 1b) and possessed the same widespread ND2 and CR haplotypes as individuals identified as Z. exasperata from other parts of the GC and PCBC, as far north as San Felipe Phylogenetic analyses and Puerto San Carlos. At the NCR locus, individuals from Bayesian inference was used to estimate phylogenetic relation- Sinaloa shared haplotypes with fish from Mulege´(Figure 2). The ships as implemented in MrBayes 3.2 (Ronquist and same was true for the nuclear gene; almost all individuals from Huelsenbeck, 2003) in which indels were not considered. Four Sinaloa possessed alleles in common with northern Z. exasperata chains were run simultaneously for 200 000 generations, using (Figure 2). This suggests that genetically indistinguishable

(a) No such pattern of genetic divergence or reciprocal monophyly was found in the nuclear marker, since the nine RAG1 alleles were less differentiated (0.18–0.73% divergence, Table 2). Nevertheless, a geographic pattern emerged from the allelic distribution. Of the two alleles found in Zapteryx from Oaxaca, one W L>W was private and the second (found in four of five fish) was shared only with organisms from the closest sampled locality (Sinaloa) and absent farther north (Figure 2). The RAG1 Bayesian phylogeny revealed a phylogeographic pattern featuring a basal poly- tomy for most northern alleles (Figure 4) and one well-supported L monophyletic clade grouping southern alleles from Sinaloa and Oaxaca (ZAPRAG1 1, 2, and 3). (b) Discussion We found that species of Zapteryx in the MP are not well charac- terized morphologically. The confusion in species’ identifications and geographic boundary demarcations of the northern banded W L

individuals, as per the mitochondrial and nuclear DNA markers Zapteryx exasperata colour polymorphism analysed in this study, manifested distinct phenotypic attributes. All individuals identified morphologically as Z. xyster are geneti- The same was true for an additional specimen identified as cally indistinguishable from Z. exasperata in mitochondrial Z. xyster from Oaxaca, although the degraded DNA could not al- (n ¼ 3) and nuclear (n ¼ 1) genes. No evidence of genetic diver- low full-length sequencing of all genes, partial sequences from gence or differentiation was found between these two groups of CR, ND2, and RAG1 matched haplotypes obtained from Z. exas- morphologically distinct fish. This pattern has been detected in perata in the GC (Supplementary Tables S2, S3, and S5), extend- other batoids. T. dumerilii and T. melaleuca were believed to be ing the distribution of this lineage much farther south than different species but morphological re-examination and analysis Mazatlan, Sinaloa. of mtDNA and nDNA indicated that T. melaleuca was a rare color variant of T. dumerilii. T. melaleuca presents unpigmented areas on the dorsal surface with normal iris pigmentation, this More than one genetic lineage of Zapteryx in the leucistic condition was attributed to mutations several genes Mexican Pacific (Donnellan et al., 2015). Similarly, coloration and skin texture Even though most of the analysed guitarfish showed only limited differentiate Raja maderensis of R. clavata; however, the lack of mtDNA differentiation (i.e. mostly shared intraspecific genetic genetic distinctiveness between them indicates that they are the variation), the five individuals from Oaxaca identified as Zapteryx same species. It is possible that nominal R. maderensis represents possessed private and divergent haplotypes (ESM CR: ZAPCR-1; a divergent population of R. clavata (Ball et al., 2016). ND2: ZAPND2-1 and ZAPND2-2; NCR: ZAPNCR-1 and Colour aberrations are not unusual in batoids; about 19 species ZAPNCR-2). These haplotypes referred hereafter as “southern” show albinism or leucism (Bigman et al., 2015). On the other haplotypes as opposed to “northern” haplotypes, were highly di- hand, the rarity of these pigmentation patterns is most likely due vergent and possessed fixed characters (Supplementary Tables to their low abundance in nature rather than their phenotypic S2–S4), in spite of the small sample size. Specifically, divergence disadvantage (Sandoval-Castillo et al., 2006). However, the colour between northern or between southern haplotypes ranged be- variation present in Z. exasperata does not correspond to any of tween <0.001 and 2.56%, whereas divergences between northern these genetic conditions. There is no complete, incomplete, im- and southern haplotypes were considerably larger, from 1.85% to perfect, or partial loss of pigments, only different patterns of col- 6.58% (Table 2). Moreover, Bayesian phylogenetic reconstruc- oration. The most conspicuous difference in coloration between tions of separate and concatenated mtDNA genes strongly sup- Zapteryx morphotypes is the presence of yellowish spots on the ported (98% bootstrap) the reciprocal monophyly of northern dorsal surface. Even though their presence appears to be geo- and southern lineages (ZAPCAT-1 and 2, Figure 3), suggesting graphically constrained to southern populations, the question re- long-standing reproductive isolation of these matrilineal lineages. mains as to what specific internal (physiological and ontological)

Table 2. Variability and genetic distance (%) of mitochondrial and nuclear genes of Zapteryx guitarﬁshes. Genetic distance among/between

Substitution Northern Southern Northern and Gene VPih/a model haplotypes þ haplotypes þ southern haplotypes Mitochondrial ND2 28 22 10 HKY* 0.10–0.41 0.31 1.97–2.50 CR 13 0 5 HKY* <0.001 to 0.33 0.00 1.85–2.02 NCR 52 40 25 TPM1ufþI ** 0.16–2.56 1. 50 4.89–6.58 CAT 93 77 33 TVMþI þG** 0.05–0.72 0.55 2.83–3.27 Nuclear Genetic distance among localities RAG1 8 2 9 HKY** 0.181 to 0.731 ND2, NADH subunit 2; CR, control region; NCR, non-coding region; CAT, concatenated; V, variable site; Pi, parsimony-informative site; h, haplotypes; a, alleles. *Akaike information criterion (AIC). **Corrected Akaike information criterion (AICc). þnorthern haplotypes refer to all haplotypes except those private to Oaxaca, which are referred to as southern haplotypes.

Figure 2. Frequency of ND2, NCR haplotypes and RAG1 alleles in guitarfishes of the genus Zapteryx from the Mexican Pacific. (sample size in parenthesis; PSC, Puerto San Carlos; PC, Punta Canoas; PSM, Punta Santa Marıa; LM, Laguna Manuela; SI, San Ignacio; BM, Bahıa Magdalena; SF, San Felipe; BA, Bahıa los Angeles; MU, Mulege´; LP, La Paz; BK, Bahıa Kino; ES, Estero El Soldado; SN, Sinaloa; OA, Oaxaca; PCBC, Pacific Coast of Baja California; GC, Gulf California; CP, Central Pacific GT, Gulf of Tehuantepec).

or external conditions (physical, chemical, and ecological) are re- supports the idea of phenotypic plasticity or adaptation associ- sponsible for this colouration. ated with environmental conditions rather than ontogenetic In the past, different life-history stages (juveniles vs. adults) changes. have been mistakenly classiﬁed as different species for several ani- The presence of secondary sexual characters is frequent in mals, because taxonomists ignored ontogenetic changes in colora- sharks and batoids. For instance, males of Squatina guggenheim tion (Booth, 1990). In some species of batoids, such as have small spines near the tips of the pectoral ﬁns (Colonello Himantura spp., juveniles feature a distinctive coloration pattern et al., 2006), and males of Dasyatis sabina have sexual dimor- from adults (Arlyza et al., 2013). Although, the different morpho- phism in dentition (Kajiura and Tricas, 1996). On the other types considered in this study could be attributed to ontogenetic hand, dorsal ocelli in Zapteryx were found in both males (40%) differentiation, our sampled organisms from Sinaloa included ju- and females (60%) in Sinaloa. veniles (40%) and adults (60%) collected by trawling and both It is likely that Zapteryx displaying alternative colour morphs possessed the same morphotype. The extent to which juvenile are adapted to different habitats. Heterogeneous environments Zapteryx possess dorsal ocelli in the southern populations is un- can act as divergent selective forces either directly on coloration known, mostly due to undersampling. However, we have exten- or indirectly on genetically correlated traits (Hubbard et al., 2010; sively collected adult Zapteryx from northern PCBC and GC Maan and Sefc, 2013). For instance, since alternative colorations populations and have not recorded the presence of ocelli. This can be inconspicuous in different habitats, coloration could be

congruent with the mtDNA signal, even though the alleles are not reciprocally monophyletic, likely from incomplete lineage sorting. Morphologically cryptic, distinct evolutionary lineages in species of batoids have been reported in the MP. In the related Rhinobatid Pseudobatus productus, haplotype frequencies differed between fish from the GC and PCBC and conformed to two divergent lineages (2.47% CR divergence (Sandoval-Castillo et al., 2004)). The same scenario of a deep mtDNA divergence was found in the golden cownose ray Rhinoptera steindachneri,in which one haplotype predominated in GC and another was found fixed in PCPBC; genetic divergence between regions was high (10.03% ND2), and GC and PCBC lineages were reciprocally monophyletic (Sandoval-Castillo and Rocha-Olivares, 2011). For Zapteryx, the main division between “northern” and “southern” lineages apparently occurs near the Gulf of Tehuantepec (Figure 2). The northern area is free year-round from the influence of the cold California Current and therefore is considered the border between the tropical and subtropical MP (Wilkinson et al., 2009). This thermal boundary is thought to reduce gene flow between marine populations (Lopez et al., 2010). However, a significant portion of the tropical ichthyofauna of the Panamanian Province extends to the mouth of the GC (Briggs and Bowen, 2012), suggesting that this thermal barrier is perme- able and possibly not enough to isolate the two lineages of Figure 3. Bayesian pylogenetic reconstruction of concatenated Zapteryx in the MP. Past climate changes could have instead mitochondrial genes (ND2, CR and NCR) of guitarfishes in the genus caused diversification of Zapteryx. The Pleistocene glacial cycles Zapteryx from the Mexican Pacific. Nodal support given as posterior caused significant alterations to marine ecosystems (Hewitt, probabilities. Morphotypes: 1 no dorsal spots, 2 with dorsal spots, 3 2000), some of which promoted lineage diversification in several new undescribed morphotype with dorsal spots and longer than marine organisms (Ludt and Rocha, 2015). The GC has been sug- wide disk. gested as a warm refuge for elasmobranchs (Sandoval-Castillo and Beheregaray, 2015) and other marine fauna (Jacobs et al., associated with foraging (Cheney, 2013) or anti-predator strate- 2004) during glaciations. Therefore, it is possible that sea level gies (Wishingrad et al., 2014). Likewise, colour differences have and temperature changes during the last glaciation period could been correlated to thermal gradients and thermoregulation effi- have isolated the two Zapteryx lineages—one in the GC (Z. exas- ciency (Trullas et al., 2007). Coloration convergence among line- perata) and other in the equatorial eastern Pacific (Z. xyster). ages experiencing similar environmental conditions has been Geographic distance also limits gene flow in sedentary benthic reported in several taxa (Manceau et al., 2010). The apparent col- species, the genetic isolation between “northern” and “southern” oration convergence between the southern lineages of Z. exasper- lineages may also have been mediated by the large geographic dis- ata and Z. xyster suggests that this trait evolved under natural tance between them, as reported in others elasmobranch with selection associated with tropical regions. Confirming the adap- similar life histories (Lewallen et al., 2007; Borsa et al., 2012). tive importance of colour morphs in Zapteryx requires different The different patterns observed in mtDNA and nDNA genes molecular approaches (e.g. ddRADseq or RNAseq) from the ones may relate to differences in their molecular evolution and to sex- used here in conjunction with oceanographic and ecological biased dispersal. The slower mutation rate of the nuclear (RAG1), approaches (e.g. seascape analysis). in comparison to mitochondrial (ND2, CR, and NCR) loci, is reflected in the significantly higher diversities of mtDNA genes. This is related to the inherently different rates of evolution of animal mtDNA and nDNA and to the hypervariability of the Genetic lineages of Zapteryx in the Mexican Pacific mtDNA non-coding regions. In addition, differences in genome The presence of undescribed cryptic lineages not only underesti- size, selection, recombination, and linkage influence the molecu- mates the diversity of elasmobranchs but also poses a challenge lar evolution of biparentally inherited (diploid) nuclear and ma- for the conservation and management of marine biodiversity. We ternally inherited mitochondrial (haploid) genes. Of particular detected two genetic lineages in the genus Zapteryx from the MP, consequence is their effective population size, which is four times which agree with the number of species described so far; however, larger in nuclear than in mitochondrial genes, making the latter phenotypic distinctions do not match the genetic lineages. Some more susceptible to random genetic drift and to more rapid line- individuals from Oaxaca provisionally named Zapteryx sp. are age sorting and fixation of haplotypes among isolated popula- different morphologically and their mtDNA is highly divergent. tions (Palumbi et al., 2001; Sclavi and Herrick, 2013). This is of In addition, these haplotypes are private and form a reciprocally particular relevance in the north–south distinction between spe- monophyletic clade from the rest. The existence of these private, cific lineages having not reached reciprocal monophyly due to in- divergent, and reciprocally monophyletic haplotypes strongly complete lineage sorting. A pattern of no nuclear and strong suggests the existence of long-term reproductive isolation consis- mitochondrial genetic differentiation may also be produced tent with different species. The nuclear RAG1 variation is by male-mediated dispersal. The strong mitochondrial

Figure 4. Bayesian phylogenetic reconstruction of RAG1 alleles of guitarﬁshes in the genus Zapteryx from the Mexican Paciﬁc. Nodal support given as posterior probabilities. Morphotypes: 1 no dorsal spots, 2 with dorsal spots, 3 new undescribed morphotype with dorsal spots and longer than wide disk.

differentiation evidenced in the mtDNA of Z. exasperata between In conclusion, a poor understanding of the patterns of mor- PCBC and GC was previously documented by Castillo-Paez et al. phological and genetic variation is mostly responsible for the (2014) and corroborated with additional data in this study confusing taxonomy, species delineation, and geographic demar- (Figure 2). This genetic signal suggests the presence of female re- cation of species’ ranges in the genus Zapteryx. While our study productive philopatry, as documented in several cartilaginous provides a significant step toward cutting through this Gordian fish (Dudgeon et al., 2012). On the other hand, the absence of nu- knot in the MP, the results point to the need of more work. clear genetic distinction revealed by the RAG1 data is suggestive Particularly fruitful would be to increase sampling effort south of of male-mediated dispersal. The extent to which the lack of ge- Sinaloa. Additional samples bridging the gap between Sinaloa and netic differentiation between PCBC and GC in RAG1 is due to a Oaxaca would help unveil the possible presence of isolation by low rate of evolution or to male-mediated dispersal requires addi- distance. We attempted to collect guitarfishes in the central and tional data from faster evolving nuclear markers (e.g. microsatel- southern MP; however, no samples were procured owing to the lites), which is currently underway and is beyond the scope of low abundance of Zapteryx in that region. Extending the geo- this paper. graphic scope of the study to Central America may also shed light on the pattern of variation of the southern lineage of guitarfishes. Taxonomic implications We found variability in Zapteryx from the MP at three scales: genetic, phenotypic, and latitudinal. However, this variation con- Supplementary data tradicts the current understanding of their classification, system- Supplementary material is available at the ICESJMS online ver- atics, and biogeography. We have discovered that (1) the most sion of the manuscript. conspicuous presumed diagnostic character of Z. xyster (i.e. dorsal ocelli) is not so; instead, it is a polymorphic character of the widespread northern phylogenetic lineage attributable to Acknowledgements Z. exasperata. Hence, a thorough morphological analysis is neces- This research was funded by internal grant 625112 from Centro sary to determine the phenotypic diagnostic characteristics of de Investigacion Cientıfica y de Educacion Superior de Ensenada Z. xyster. (2) Z. exasperata may be found as far south and Z. xyster (CICESE) to ARO. The first author benefited from a fellowship may be found as far north as Oaxaca. However, this needs further from CONACyT to carry out her PhD in Marine Ecology at corroboration given our limited number of samples from the CICESE. We thank E. Onate~ Gonzalez, O. Santana Morales, southern MP. (3) At least two independent species-specific P. Suarez Moo, F. Cicala, G. Andrade Dominguez, and A. Torres mtDNA lineages occur in the MP, the northern lineage corre- Torres for assistance with fieldwork. We thank D. Chavez sponds to Z. exasperata and the southern may correspond to Arrenquın for the collection of individuals on INAPESCA cruises Z. xyster. (4) The coloration and genetic patterns observed in fish in Sinaloa. We thank I. Hernandez Candelario and F. J. Ponce for from Sinaloa (locality 7) are consistent with the possible hybridi- help in figure preparation. F.G.M. thanks to Instituto Politećnico zation between females GC Z. exaspertata and southern males Nacional (EDI, COFAA fellowships). Two anonymous reviewers Z. xyster, such that only nDNA is being introgressed in this provided constructive comments that helped improve the contact zone. manuscript.

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