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 Polite´cnico 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 pat- terns 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 gui- tarfish 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 at- tributable 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 taxo- nomic 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. VC International Council for the Exploration of the Sea 2017. All rights reserved. For Permissions, please email: [email protected] Downloaded from https://academic.oup.com/icesjms/article-abstract/74/6/1630/3061538/Cutting-through-the-Gordian-knot-unravelling by Universidad Nacional Autonoma de Mexico user on 01 September 2017 Variation patterns in Mexican Pacific Zapteryx 1631 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 (Mendonc¸a 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 ge- netic 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 (Mendonc¸a 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.