Molecular Identification of Ichthyoplankton in Cabo Pulmo National Park 1 Using molecular identification of ichthyoplankton to monitor 2 spawning activity in a subtropical no-take Marine Reserve 3 4 5 6 Ana Luisa M. Ahern1, *, Ronald S. Burton1, Ricardo J. Saldierna-Martínez2, Andrew F. Johnson1, 7 Alice E. Harada1, Brad Erisman1,4, Octavio Aburto-Oropeza1, David I. Castro Arvizú3, Arturo R. 8 Sánchez-Uvera2, Jaime Gómez-Gutiérrez2 9 10 11 12 1Marine Biology Research Division, Scripps Institution of Oceanography, University of California 13 San Diego, La Jolla, California, USA 14 2Departamento de Plancton y Ecología Marina, Centro Interdisciplinario de Ciencias Marinas, 15 Instituto Politécnico Nacional, CP 23096, La Paz, Baja California Sur, Mexico 16 3Cabo Pulmo National Park, Baja California Sur, Mexico 17 4The University of Texas at Austin, Marine Science Institute, College of Natural Sciences, 18 Port Aransas, Texas, USA 19 20 21 22 23 24 25 *Corresponding author: [email protected] 1 Molecular Identification of Ichthyoplankton in Cabo Pulmo National Park 26 ABSTRACT: Ichthyoplankton studies can provide valuable information on the species richness 27 and spawning activity of fishes, complementing estimations done using trawls and diver surveys. 28 Zooplankton samples were collected weekly between January and December 2014 in Cabo 29 Pulmo National Park, Gulf of California, Mexico (n=48). Fish larvae and particularly eggs are 30 difficult to identify morphologically, therefore the DNA barcoding method was employed to 31 identify 4,388 specimens, resulting in 157 Operational Taxonomic Units (OTUs) corresponding 32 to species. Scarus sp., Halichoeres dispilus, Xyrichtys mundiceps, Euthynnus lineatus, 33 Ammodytoides gilli, Synodus lacertinus, Etrumeus acuminatus, Chanos chanos, Haemulon 34 flaviguttatum, and Vinciguerria lucetia were the most abundant and frequent species recorded. 35 Noteworthy species identified include rare mesopelagic species such as the giant oarfish 36 (Regalecus glesne) and highly migratory and commercially important species such as black 37 skipjack (Euthynnus lineatus) and yellowfin tuna (Thunnus albacares). Spawning activities 38 showed distinct seasonal patterns with the highest abundance of ichthyoplankton recorded during 39 spring, highest species richness during summer (90 OTUs) and lowest species richness during 40 winter (28 OTUs). A total of seven OTUs were recorded throughout the year (4%), 11 OTUs 41 during three seasons (7%), 36 OTUs in two seasons (23%) and 106 OTUs were recorded in only 42 one season (66%). The study found eggs and/or larvae of 47 species that were not previously 43 reported in Cabo Pulmo National Park. Results allow resource managers to compare shifting 44 populations and spawning patterns of species that may be affected by both conservation efforts 45 and broader oceanographic changes associated with climate change. 46 47 48 KEY WORDS: Marine protected area · DNA barcoding · molecular ecology · marine 49 conservation · Cabo Pulmo National Park · Gulf of California · Mexico 2 Molecular Identification of Ichthyoplankton in Cabo Pulmo National Park 50 INTRODUCTION 51 52 Cabo Pulmo National Park is a subtropical no-take marine reserve located on the 53 southeast coast of Baja California Sur, Mexico in the Gulf of California (Fig. 1A, B). This 54 national park is a unique example of a successfully managed marine protected area (Aburto- 55 Oropeza et al. 2011). Established as a Mexican national marine park in 1995, Cabo Pulmo 56 National Park is recognized as a UNESCO World Heritage Site. Since 1995, the community of 57 Cabo Pulmo voluntarily chose to expand the no-take zone from an initial 35% to nearly 100% of 58 the 27-square mile park (Aburto-Oropeza et al. 2011) (Fig. 1B). Although the community of 59 Cabo Pulmo already supported a small tourism industry, it was bolstered as the biomass, 60 abundance and diversity of charismatic and commercially important fish species as well as 61 marine mammals increased. A ten-year study showed a 463% increase in total fish biomass and 62 a 1070% increase in biomass of top predators since 1995, the largest ever measured in a marine 63 reserve worldwide (Aburto-Oropeza et al. 2011, 2015). 64 While the biota of the rocky and coral reefs of Cabo Pulmo National Park are thriving, 65 they are still vulnerable to a multitude of threats including coastal development (Arizpe & 66 Covarrubias 2010), overfishing (Johnson et al. 2017), and climate change (Verutes et al. 2014, 67 Robinson et al. 2013, 2016). As the area has garnered more public and academic attention, large 68 international developers have proposed development projects in neighboring communities that 69 could have negative effects for Cabo Pulmo National Park’s coastline and marine biota (Arizpe 70 & Covarrubias 2010). As a consequence of global climate change, it is predicted the oceans will 71 experience a significant increase in sea surface temperature in the next 100 years (Levitus et al. 72 2009), which could result in more frequent and intense El Niño Southern Oscillation (ENSO) 3 Molecular Identification of Ichthyoplankton in Cabo Pulmo National Park 73 events, many of which have had significant effects on the region in the past and recent years 74 (Timmermann et al. 1999, Robinson et al. 2013, 2016). Since 2010 the Gulf of California has 75 experienced an increase in sea surface temperature and decrease of wind speed, resulting in 76 lower sea surface chlorophyll-a concentration than previous years (Robinson et al. 2016). The 77 waters of Cabo Pulmo National Park will likely face increased fluctuations in temperature, 78 dissolved oxygen concentrations, pH, nutrient content, and circulation that could negatively 79 impact this delicate rocky and coral reef ecosystem (Doney et al. 2012). 80 Careful monitoring of vulnerable coastal marine ecosystems is needed to track biotic 81 changes that may occur as a result of a changing climate and to inform marine resource 82 management decisions (Harada et al. 2015). The abundance and species composition of 83 ichthyoplankton collected from the water column provides valuable information concerning the 84 broadcast spawning activities of fishes and plays a significant role in the assessment and 85 management of marine ecosystems (Gleason & Burton 2012). Ichthyoplankton surveys can be 86 used as a fisheries independent indicator of ecosystem health, estimating species-specific 87 spawning biomass, reproductive periods, overall reproductive strategies and population 88 dynamics as a function of environmental variability (Lo 2001, Aceves-Medina et al. 2003, 2004). 89 They can also help identify the location of critical spawning habitat that should be protected in 90 order to ensure the present and long-term sustainability of vulnerable fish populations (Sala et al. 91 2003). 92 Historically, scientists and fisheries managers have relied on morphological identification 93 of ichthyoplankton (mostly larvae, occasionally eggs) to determine which species spawn in a 94 particular area (Ahlstrom & Moser 1980, Aceves-Medina et al. 2003, 2004, Miller & Kendall 95 2009, Harada et al. 2015). A considerable drawback to this morphological method is the 4 Molecular Identification of Ichthyoplankton in Cabo Pulmo National Park 96 difficulty of telling species apart at early life stages; in fact, many species have virtually 97 indistinguishable eggs and successful identification of fish eggs and preflexion fish larvae using 98 morphological characteristics alone requires years of study (Ahlstrom and Moser 1980, Hyde et 99 al. 2005). Even then, morphological experts can experience high uncertainty in fish egg 100 identification (Ahlstrom & Moser 1980, Moser et al. 1974, 1993), which can prove costly when 101 these data are used to determine population abundance and make management decisions for 102 targeted species. Where traditional morphological analysis may have difficulty distinguishing 103 species with similar morphological characteristics, molecular genetic analysis can provide 104 accurate species identification to infer spawning strategies and determine the magnitude of 105 reproductive efforts of fish assemblages (Burton 2009, Harada et al. 2015). 106 Molecular analysis of ichthyoplankton provides valuable information about temporal and 107 geographic spawning activity and can be used for the purposes of stock assessments and 108 monitoring ecosystem health (Perez et al. 2005, Harada et al. 2015). The use of molecular 109 genetic tools to assist in conservation efforts is becoming increasingly affordable (NHGRI 110 Genome Sequencing Program, http://www.genome.gov/sequencingcosts/). Other methods of 111 monitoring fish populations include diver-conducted monitoring surveys, which until now have 112 been the primary source of information about the abundance and diversity of fish species found 113 in Cabo Pulmo National Park’s waters (Alvarez-Filip et al. 2006, Aburto-Oropeza et al. 2011, 114 2015). Many species reproduce at night when divers are unable to observe fish spawning events 115 (Claro & Lindeman 2003, Erisman et al. 2014). Trawling is not only invasive to vulnerable fish 116 populations and sensitive marine environments, but are often size and species selective. 117 Additionally, trawling surveys are not allowed in most marine protected areas, making this 118 method generally unsuitable. Video assessments can also be biased depending on the locations 5 Molecular Identification of Ichthyoplankton in Cabo Pulmo National Park