Reproductive patterns in demersal crustaceans from the upper boundary of the OMZ off north-central Chile

Author’s Accepted Manuscript REPRODUCTIVE PATTERNS IN DEMERSAL CRUSTACEANS FROM THE UPPER BOUNDARY OF THE OMZ OFF NORTHCENTRAL CHILE María de los Ángeles Gallardo, Andrés E. González López, Marcel Ramos, Armando Mujica, Praxedes Muñoz, Javier Sellanes, Beatriz Yannicelli

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To appear in: Continental Shelf Research Received date: 5 January 2017 Revised date: 21 April 2017 Accepted date: 28 April 2017 Cite this article as: María de los Ángeles Gallardo, Andrés E. González López, Marcel Ramos, Armando Mujica, Praxedes Muñoz, Javier Sellanes and Beatriz Yannicelli, REPRODUCTIVE PATTERNS IN DEMERSAL CRUSTACEANS FROM THE UPPER BOUNDARY OF THE OMZ OFF NORTH-CENTRAL CHILE, Continental Shelf Research, http://dx.doi.org/10.1016/j.csr.2017.04.011 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 galley proof before it is published in its final citable 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.

REPRODUCTIVE PATTERNS IN DEMERSAL CRUSTACEANS FROM THE UPPER BOUNDARY OF THE OMZ OFF NORTH-CENTRAL CHILE

María de los Ángeles Gallardo1,2, Andrés E. González López1,2,3,4,5,6,7, Marcel Ramos3,5,2,7, Armando Mujica4, Praxedes Muñoz3,2., Javier Sellanes2,3,5, Beatriz Yannicelli2,3,5,6 1

Programa de Doctorado en Biología y Ecología Aplicada, Universidad Católica del Norte y Universidad de La Serena. 2

Centro de Estudios Avanzados en Zonas Áridas, Facultad de Ciencias del Mar, Universidad Católica del Norte, Campus Guayacán, Larrondo 1281, Coquimbo, Chile. 3

Departamento de Biología Marina, Facultad de Ciencias del Mar, Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile. 4

Departamento de Acuicultura, Facultad de Ciencias del Mar, Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile. Núcleo Milenio “Ecología y manejo sustentable de islas oceánicas”, Universidad Católica del Norte, Facultad de Ciencias del Mar, Coquimbo, Chile. 5

6

Centro Universitario Regional Este, Rocha, Universidad de la República, Uruguay.

7

Centro de Innovación Acuícola Aquapacífico, Coquimbo, Chile.

ABSTRACT Pleuroncodes monodon (Crustacea: Munididae) supports one of the main trawling fisheries over the continental shelf off Chile between 25°S and 37°S within the upper boundary of the oxygen minimum zone (OMZ). Although the reproductive cycle of P. monodon has been described, the relationship between this key biological process and the variability of the OMZ has not been comprehensibly addressed neither for P. monodon nor for other OMZ resident species. In this study a set of 14 quasi-monthly oceanographic cruises carried out between June 2010 and November 2011 were conducted over the continental shelf off Coquimbo (30ºS) to investigate the temporal variability of: i) dissolved oxygen concentration, temperature and chlorophyll-a at relevant depths ii) the presence and proportion of occurrence of P. monodon ovigerous females and juveniles from benthic trawls; iii) the presence of different stage larvae in the plankton, and iv) similar biological data for other species from the OMZ and shallower depths crustaceans. During summer months oxygen levels and bottom temperature were lower than in winter, while chlorophyll-a concentration was maximum in summer coinciding with an active (but not maximum) upwelling season. P. monodon maximum egg carrying occured in winter during periods of increased oxygenation. Egg

carrying females were never found at depths where oxygen concentration was below 0.5 ml L-1, while over 50% of the autumn and spring cohorts of juveniles occurred at oxygen concentrations below that level. The depth range occupied by ovigerous females was more restricted than the rest of the population and their depth of occurrence followed the variability of the upper OMZ. The larval release period of OMZ resident species extends over late winter and spring, and its main peak precedes that of coastal species (spring) and the spring-summer chlorophyll-a maximum. We propose that for OMZ resident species, brood carrying during warmer and more oxygenated conditions in the adult benthic environment, might favour embryonic development, so OMZ seasonal variability could be acting as a selective pressure to synchronize reproductive periods.

Key words: Pleuroncodes monodon, Oxygen Minimum Zone, reproductive cycle, larval release, Humboldt currents systems.

1 INTRODUCTION The Humboldt Current System (HCS)in the South Pacific Ocean (SPO), is one of the most productive in the world oceans (Gilly et al., 2013), it is characterized by the presence of subsurface waters with low dissolved oxygen that intrude over the continental shelf to very shallow depths. Naturally occurring bodies of water with oxygen concentrations below 0.5 ml L-1 are known as Oxygen Minimum Zones (OMZs) (Levin 2003; Helly and Levin 2004;Paulmier and Ruiz-Pino 2009; Ulloa and Pantoja 2009). OMZs constitute a physical/chemical barrier for many species of aerobic metazoans (Childress and Seibel, 1998) that cannot attain their minimum metabolic demands in low oxygen concentrations (Apablaza and Palma, 2006; Antezana, 2009; Escribano et al., 2009). Therefore, the presence and annual variability of the OMZ on the continental margins of western South America influences benthic and pelagic communities (Fuenzalida et al., 2009). The variation of dissolved oxygen concentration with depth together with depth itself are the main factors underlying the change in benthic megafauna composition and abundance across the continental shelf and slope off central Chile (36ºS) (Quiroga et al., 2009).The pelagic vertical distribution and migration of several planktonic species (such as Calanus chilensis and Subeucalanus crassus) is constrained at depths above the OMZ (Escribano et al., 2009). The temporal variability of oxycline depth and OMZ intensity might also play a role in the modulation of life traits like reproductive cycles of tolerant aerobic species since gonad maturation and egg carrying (in crustaceans) are particularly demanding processes in terms of energy (Fernandez et al., 2000, Erikson et al. 2006). For benthic adults, meeting the energetic cost of reproduction could depend on oxygen availability; for larvae, vertical migration as a strategy to avoid offshore

advection during peak upwelling periods, depends not only on swimming capacity but also on low oxygen tolerance (Yannicelli et al, 2006). The influence of oxygen availability on reproductive cycles of pelagic and benthic organisms in the HCS has recently been addressed. For example, Eucalanus inermis, a copepod that migrates daily intruding OMZ waters during daytime, remains above the OMZ during the reproductive season (Hidalgo et al., 2005; Escribano et al., 2009; Fernández et al. 2002) . In the Eastern South Pacific (ESP) the OMZ is associated with the Equatorial Subsurface Water, a water mass that is transported from the equator to about 42°S (Silva et al., 2009) by the Perú-Chile Undercurrent (PCU). PCU variability is largely forced remotely by equatorial propagating perturbations. Low oxygen waters flow along the slope throughout the year following PCU variability, but their presence on the adjacent continental shelf also depend on local physical and biogeochemical processes. Meridional advection in the Concepción shelf area (37°S) would be the main driver of seasonal oxycline depth variability: shallow (< 30m depth) during spring-summer months and deep (>80m) during autumn-winter(Paulmier et al., 2006;Fuenzalida et al., 2009; Charpentier et al., 2012). A similar seasonality has been observed at 30°S although forcing mechanisms are not clear yet (Charpentier et al., 2012). Because the temporal variability of the OMZ along the continental margin between 30°S and37°S is influenced by remote and local processes, inter-annual (ENSO) and synoptic (local winds) scales of variability also have important signatures (Charpentier et al., 2012). There is little information available on how seasonal variability on oxygen concentration in benthic ecosystems can influence the reproductive patterns of macroinvertebrates. It is important to understand the relationship between temporal variability in the OMZ and biological and ecological attributes of the species that live within this environment, such as their reproductive cycles. This information would contribute towards an understanding of population dynamics and responses to inter-annual environmental variability. Nevertheless, knowledge on the physiology of species that inhabit the OMZ and its links to characteristic environmental patterns in these areas is limited. The squat lobster Pleuroncodes monodon is an important bentho-pelagic crustacean species in the continental margin of the EPS. P. monodon adult habits vary with latitude: pelagic populations above the oxycline occur between 12-18°S (Gutierrez et al, 2008), but fully benthic adults occur throughout the Chilean continental shelf between 25-37°S in association with the OMZ (Bahamonde et al 1986). Benthic populations comprise several generations and are among the main targets of trawl fisheries in Chilean continental shelf (Andrade, 1986). Although this fishery has been subject of various management strategies since the late 1970’s, populations of the exploited

benthic P. monodon (29-30º and 36-37°S) have shown strong inter-annual variability in biomass, distribution and recruitment (Bahamonde et al., 1986). Associations between reproductive patterns (period of egg carrying and larval release) and seasonal variation in the oceanographic conditions are still unknown. Improving our knowledge on this subject will thus allow us to better understand the inter-annual variability in populations, and to evaluate the potential effects of projected climate change scenarios. Available biological information on Pleuroncodes monodon is derived from fisheries surveys, scientific surveys and laboratory experiments, namely that: (1) egg carrying females occur from April to November (Palma and Arana, 1997) and there may be more than one brood annually per female under laboratory conditions(Thiel et al., 2012), (2) the planktonic phase of P. monodon includes five larval zoeas stages (Fagetti and Campodonico, 1971), one megalopa and one juvenile. (3) Megalopas are present in the plankton from mid-spring to late summer. Juveniles become fully benthic in March and April (during the late austral summer to early autumn) (Gallardo et al., 1994; Roa et al., 1995). Megalopas and juveniles migrate extensively between the surface and the hypoxic zone (Palma, 1994; Yannicelli et al., 2012). Nevertheless, no integrated studies have been carried out associating the annual variability of oceanographic conditions with the annual cycle of benthic reproduction and pelagic larval occurrence. In this study, the period of egg carrying and the presence of early larvae in the plankton are investigated for Pleuroncodes monodon as well as for other crustacean species trawled at 30ºS, and compared with those of shallower species. We hypothesized that. i: the spatio-temporal distribution of ovigerous P. monodon present is associated with higher dissolved oxygen levels than the rest of the benthic population. ii: the annual cycle of larval release centered in late winter-early spring is a common feature of crustacean species associated with the upper OMZ. iii: Larval release timing of OMZ crustacean species is not in phase with that of species living at shallower depths. In addition, we propose a comprehensive conceptual model for the seasonal egg-carrying period and larval release of upper OMZ residents with annual environmental cycles (oxygen concentration, Chlorophyll). 2 METHODS 2.1 Reproductive seasonality of Pleuroncodes monodon and seasonal variability of the OMZ In order to evaluate the reproductive seasonality of Pleuroncodes monodon at 30°S and the seasonal variability of the OMZ, information was directly taken in the field and was compiled with literature

providing information on the temporal variability of: i) the presence and proportion of occurrence of ovigerous females in the area of Coquimbo; ii) the presence of larvae in the plankton and details of the larval stage (the period of larval release); iii) presence of juveniles benthic samples and iv) dissolved oxygen concentration, temperature and Chlorophyll-a at relevant depths (to characterized the OMZ and trawling depths, see 2.2.1). The observations were made within the SIPO INNOVA 07CN13 IXM-150, during of which a set of 14 quasi-monthly oceanographic cruises were carried out between June 2010 and November 2011 (Table 1, Figure 1). On each cruise, bottom samples were taken with a modified Agassiz trawl at stations 5, 6, 7, 8, 10, 11, 14 and 16 (Figure 1), between depths 50 to 250 m. The sample was sorted to record total nonfemales, ovigerous females, males and juveniles, and the data was standardized to individuals per m-2. Juveniles were also measured from the base of the rostrum to the posterior margin of the carapace (standard measurement of carapace length CL) (Kato, 1974). Plankton samples were taken at stations 1, 3, 5, 8, 9, 11, 14, 16, and 18, with a Hydro-Bios Multinet (0.25m2 frame and 200 μ mesh size), composed of 5 nets with integrated flowmeters and an automatic electronic mechanism for opening and closing. The samples obtained were fixed in a solution of neutralized formalin in sea water (5%). Sample processing is described below (larval release). On each cruise, CTD-O profiles were taken at each station (SeaBird 19 plus) and temperature data was also measured. Additionally water samples were taken at station 1 to measure the dissolved oxygen concentration, using the Winkler titration method using a Titrino-plus (Metrohom) automatic titrator, and Chlorophyll-a (Chl-a) was also measured using an spectrophotometric method following Strickland and Parsons (1972). 2.1.1 Hydrography of the study area In order to characterize the seasonal variation in temperature (°C) and oxygen concentration (ml L1

) time series for these variables were constructed from station 1 (Figure 1) at 50, 100, 150, 250 and

500 m. This station was chosen because the vertical variability of temperature and dissolved oxygen correlated with that of the entire study area and was deep enough to cover the entire depth range distribution of P. monodon. The hydrographics data were processed and analyzed using Matlab 12. 2.1.2 The monthly proportion of ovigerous females, females and juveniles From each trawl sample, P. monodon females were recognized by their sexual characteristics: lack of first pleopods (present in males); position of gonopores in third pairs of periopods (males: first pleopods); pleopods 2-5 unirramous with long setae to which eggs are attached (males: pleopods 3-

5 reduced) (Baba et al., 2011; Thiel and Lovrich, 2011). Individuals were defined as juveniles when no sexual characteristics were distinguished. The depth, surface chlorophyll and bottom temperature and oxygen where P. monodon individuals were captured at each station, were used to evaluate bio-physical associations. Cumulative frequencies of the presence/absence of ovigerous females, females, males and juveniles as a function of bottom dissolved oxygen were constructed. The average abundance of females per month was estimated based on a delta distribution and expressed as individuals m-2(Pennington, 1983). The proportion of ovigerous females (OF) and non-ovigerous females (NOF) was estimated for cruise and depth (between June 2010 and November 2011). 2.2

Larval release

To assess the period of larval release, we followed the abundance of zoea I in the plankton throughout the study period. The period of larval release was also evaluated for five additional species: the armed box crab Platymera gaudichaudii, which inhabits the OMZ as P. monodon, a subtidal species (Neotrypaea uncinata) and three coastal species (Emerita analoga, Lepidopa chilensis and Blepharipoda spinimana) (Table 2). The different species and larval stages were determined under a Carl Zeiss stereoscopic microscope from 10X to 80X, and a 2X magnifier and quantities were expressed as the number of individuals100 m-3 at each station. The monthly abundance estimates were based on a delta distribution (Pennington, 1983), which allows for the consideration of zero abundances, as irregular distributions are characteristic of the plankton. The abundance of zoea I within each species should be able to provide an approximate estimate of the main release period. 2.3 Statistical analysis A generalized linear model (GLM) analysis with a binomial distribution was used to investigate the influence of bottom oxygen concentration (DO ml L-1), bottom temperature (T °C), depth and Chlorophyll-a (5m depth)on the presence (1) or absence (0) of ovigerous females, non- ovigerous females and juveniles on each SIPO cruise. The variables were integrated in multiplicative logistic regression, where we tested the significance of both additive and multiplicative terms. The best model was chosen after backward elimination of less significant terms using Package lme4 (version 1.1-12, functions “glm” and “step”), minimizing the Akaike Information Criterion (AIC) (Zuur et al., 2009). Also, the explained deviance (D2 o Pseudo-R2) for the model was estimated (Zuur et al., 2009). All statistical analyses were carried out using the R software (R Core Team, 2015).

3 RESULTS 3.1 Hydrography 2010-2011 The variability of the position of the OMZ co-varied in and outside the bay (as seen in Figure 2 a, b): shallower (summer) or deeper (winter).Throughout the entire sampling period the oxygen concentration at the time series station 1 remained below 1 ml L-1 at depths ranging from 50 to 250 m (Figure 2c and 2d) which highlights the influence of the OMZ within this coastal area. The greatest fluctuations in oxygen concentration were observed at 50 m (0.4 −4.3 ml L-1) and 500 m (0.8−2.9 ml L-1). During winter the oxygen concentrations were higher than in summer at all depths. The range of variation of oxygen concentration at 100 and 250m was 1.3 ml L-1 and 0.16 ml L-1, respectively; and followed the same seasonal pattern as previously described for 50 and 500 m. At 50 m and 100 m depth, important fluctuations were also observed within seasons (Figure 2c). A seasonal fluctuation was also observed for temperature at 50, 100, 150 and 250 m. The greatest fluctuations in temperature (>2 °C) were observed at 50 m (11.1−13.1 ºC) and 250 m (9.27−11.35 ºC). The highest temperatures were recorded in 2011 at all depths during autumn-winter, similarly, the temperatures recorded in June 2010 were higher than those recorded in spring and summer months. The amplitude of temperature fluctuation was 2ºC at 100 and 150 m depth, following the same seasonal pattern as previously described (Figure 2e). Chlorophyll-a concentration in surface waters was highest during summer months. A progressive increase in Chl˗a concentration from spring (October 2010; November 2011) was observed until mid-summer (at 0, 5 and 10m depth) (Figure 2f). The variation in Chl˗a concentration ranges from 9.911 to <1 mg m-3 at the surface, with a similar seasonal pattern observed at 5 m. During autumn and winter months concentrations of Chl˗a were <1 mg m-3. 3.2 Egg carrying period During the SIPO cruise ovigerous females were found at depths of 50, 100, 150 and 250 m. The proportion of ovigerous females was higher during autumn-winter than in the other seasons) (Figure 3a) and reached 100% in August 2010 and August 2011. Between October 2010 and March 2011 the proportion of ovigerous females was less than 10%, and their abundance was below 5 individuals per m-2. From March 2011 the proportion of females begins to progressively increase peaking in August and then declining in October 2011.

Ovigerous females were usually found in a narrow depth range (except august 2011). They occupied shallow sediments (50 m) during spring and summer months moving to deeper stations during autumn-winter (>100) (Figure 3b). Non-ovigerous females were distributed over a wider depth range than ovigerous females, especially during spring-summer months (Figure 3b, c). For dates when both ovigerous and non-ovigerous females were sampled, their depth distribution overlapped at 50 or 100m depth stations with bottom oxygen levels above1 ml L-1. Overall, the benthic stages of P. monodon were found at oxygen levels that ranged from 0.16 to 3.04ml L-1and temperatures (T) from 9.5°C to 12.31°C. Nevertheless non-ovigerous females occurred at DO above 0.16 ml L-1 and T above 9.5°C, while ovigerous females only occurred at DO above 0.5 ml L1 and T above 10.15°C. The 50% of ovigerous females were found at DO in the range of 0.5 to 1 ml L-1.While below 1mlL-1DO, 69% of males, 75% of non-ovigerous females and 91% of juveniles were recorded. For these stages 50% of the population was found below DO concentrations of 0.72, 0.55 and 0.45 ml L-1respectively (Figure 4). Dissolved oxygen and Chlorophyll-a were the main variables that explained the occurrence of ovigerous females (GLM Best model p<0.05, Table 3). The probability of finding ovigerous females significantly increases (p = 0.0161) with increasing oxygen concentration (within the range of DO of the species).Contrariwise, a significant negative relationship was found between the frequency of ovigerous females and chlorophyll-a (p = 0.0350). 3.3 Larval abundance and release From the six species of decapod crustacean larvae identified (Table 2), the larvae of P. monodon and N. uncinata were the most abundant. During June 2010 and between March and May 2011 the larvae of these six species were either absent or poorly represented in the plankton (except for the larvae of Emerita analoga) (Figure5). There is a time-lag in temporal patterns of larval abundance between coastal species (E. analoga, B. spinimana and L. chilensis) and OMZ residents (P. gaudichaudii and P. monodon). The three coastal species analyzed were generally representative of sandy beaches. The residence period of B. spinimana and L. chilensis larvae was limited to the spring-summer and mainly concentrated at coastal stations (Figure. 1b stations 8, 9 and 11). Zoea I of B. spinimana and L. chilensis occur at higher numbers in the plankton during the spring-summer (Figure5) and their advanced stage of development increased their abundance in the late summer at coastal stations. E. analoga zoea I were observed throughout the majority of the cruises and at least 3 peaks with

similar abundance (~100 larvae m-3) were identified for this development stage, in spring-summer (2010 and 2011) and autumn 2011. Zoeas I of OMZ resident species (P. monodon and P. gaudichaudii) were more abundant during austral winter than summer. This contrasted with observations of coastal species larval abundances that increased during spring (Figure5). Zoeas I of P. monodon were collected throughout the year. However, the main period of larval release (presence of Zoeas I) was observed in austral winter 2011 (June- August), with a peak in June 2011 (>600 larvae 100 m-3).The abundance of Zoeas I larvae of P. monodon decreased progressively until November, despite the fact that ovigerous females were still observed during November. The advanced stages of this species were only found during summer (December 2010 and January 2011), mainly in the stations further away from the coast (station 5 and 18) and they were absent in October and November 2011. Similar to P. monodon, the Zoeas I larvae of P. gaudichaudii (a resident species of the OMZ) were found on all cruises. However two peak abundances were recorded with the first peak in August 2011 (winter) and the second in November 2011 (spring). Advanced stages and megalopa of P. gaudichaudii were found during the summer months in oceanic stations, similar to P. monodon. N. uncinata larvae displayed a similar pattern to P. gaudichaudii, Zoeas I increased in abundance in August 2011 (~ 300 larvae100m-3), decreasing during spring. For both OMZ resident species and N. uncinata, the advanced stages were first observed in October 2010, but for the same period in 2011 these stages were absent. The peak of coastal species larval abundance during spring-summer (2010-2011) coincided with the maximum Chlorophyll-a concentrations. These peaks in larval abundance were not present in the case of OMZ resident species. Contrarily, E. analoga larval abundance peaked in autumn, differing from all other species. OMZ resident species and subtidal N. uncinata release their larvae between 1−3 months before coastal species (Figure5) and it is not related to the period of maximum surface productivity, which occurs well after the main period of larval release. 3.4 Juveniles of Pleuroncodes monodon: The juveniles of P. monodon were found on the benthos mainly during two seasons of the year, spring (187 ind. m-2) and autumn (1407 ind. m-2). The major peak in juvenile abundance was recorded in the autumn during May 2011 (Figure 6). The juveniles were found at all depths, mainly below 100 m. Because the minimum carapace length of juveniles of spring and autumn peaks were similar, and maximum lengths were larger in spring, we determined that two different cohorts overlap during spring months. It is assumed that each cohort is the product of larvae released in the

winter and spring, respectively (Figure 6). The occurrence of juveniles coincides with the decrease of surface Chlorophyll-a. Juveniles were found at oxygen levels that ranged from 0.16 to 3.04 ml L1

and T 9.5 to 12.31 ml L-1. Although 50% of the occurrence of juveniles was registered under 0.5

ml L-1, DO did not have a significant effect on the occurrence of this stage. Depth was the variable that better explained the occurrence of juveniles (GLM Best model, Table 3). The probability of finding juveniles significantly increased (p = 0.0371) with depth down to 250 m at 30°S. 4 DISCUSSION 4.1 Seasonal OMZ variation Seasonality of the OMZ was evident from the fluctuation of oxygen in the water column recorded between 2010 and 2011. The more oxygenated water column during winter in comparison with summer is similar to the seasonality described by Charpentier et al. (2012), after evaluating 10 years of monthly observations on the continental shelf off Concepcion (37ºS). The observed thermal pattern also agrees with the data reported by Charpentier et al. (2012), where the warmer periods, in the deeper layers, coincide with more oxygenated periods. The high variability of oxygen in the first 100 m depth has also been observed at 37 ° S where it has been associated with local winds forcing(Pizarro et al., 2016). In particular, the low oxygen concentrations at 50 and 100 m in April 2011 we found for the Coquimbo area was preceded by a week of southerly winds (www.ceazamet.cl), which are favorable for coastal upwelling and the rise of subsurface waters (www.ceazamet.cl). 4.2 The effect of temperature and oxygen on egg carrying in Pleuroncodes monodon At ~30ºS the observed egg carrying period (seasonality and length) in P. monodon is consistent with that reported by Palma and Arana (1997) for ~37°S. Furthermore the peak of ovigerous females during winter agrees also with previous reports for the Coquimbo region ~30°S (EspinozaFuenzalida, 2008). The period of maximum egg carrying in winter, and the location of females on the benthos, is coincident with periods of increased oxygenation, and it is consistent with the initial hypothesis. Ovigerous females presented a higher dissolved oxygen threshold (0.5 ml L-1) than the rest of P. monodon benthic population (0.16 ml L-1) (Figure 3 and 4). This difference resulted in a reduction of the depth range that ovigerous females occupied as compared with the rest of the population, as well as in seasonal depth variability (deeper in winter and shallower in spring-summer), following

the bottom DO trends. Although seasonal bathymetric variability has been described previously for females of P. monodon (Bahamonde et al., 1986), this is the first time that it is associated with threshold levels of bottom DO. We propose that the seasonal bathymetric migration of ovigerous females, observed in this study and previously described by Bahamonde et al. (1986), is related to the search for oxygen concentrations favoring embryonic development. Additionally the spatial separation between ovigerous females from the other benthic life stages (and sex) in association with this environmental variable had not been previously proposed, and further supports the relevance of DO on shaping the life cycle of the benthonic species along the Humboldt Current System (see below). A higher dissolved oxygen threshold for the distribution of ovigerous females may be due to either of both of the following reasons: i) increased metabolic demand of females during egg carrying that could not be met at extremely low environmental oxygen levels; ii) a potential increase in egg mortality or developmental delay due to hypoxia. Decapod crustaceans invest important quantities of energy to sustain the active behaviour of parental care and to maintain a stable partial pressure of oxygen inside the brood (Fernandez et al., 2000). This type of active behaviour by decapods is strengthened when egg development occurs in hypoxic environments (Eriksson et al., 2006).On the other hand, even in organisms adapted to hypoxic environments, such as Nephrops norvegicus, the survival rate of eggs in advanced stages decreases significantly or early hatching occurs at very low oxygen concentrations (Eriksson et al., 2006). During the SIPO cruise it was observed that ovigerous females were mainly found during more oxygenated periods, at depths where the water temperature was >11°C, one degree higher than the spring-summer temperature (hypoxic period), increasing to >12°C during the peak of the egg carrying period (June to August). Temperature plays an important role in the rate of embryonic and larval development, as temperature increases the developmental rate also increases in several crustacean taxa, including in P. monodon (Fagetti and Campodonico, 1971). As the rate in embryonic development increases, egg carrying time is reduced (Anger, 2001; Anger et al., 2003). Moreover it has a beneficial effect on the survival rate of newly hatched Zoeas I (Yannicelli and Castro, 2013). Both variables (temperature and oxygen) observed during the egg carrying stage could present a window of opportunity for egg carrying and embryonic development. Furthermore, changes in the bathymetric position of ovigerous females (in contrast to non-ovigerous females, Figure 3) observed in this study, and also previously described in the Bay of Concepción (Bahamonde et al., 1986;

Palma and Arana, 1997), could be due to the pursuit of a suitable temperature and oxygen conditions in response to the penetration of the OMZ onto the platform as the spring approaches. 4.3 Larval release In spite of finding Zoea I during most of the year two main larval release picks were recorded. The principal larval release of residents of the ZMO in winter, and the second peak of abundance found in P. gaudichaudii was consistent with that modeled by Yannicelli et al. (2012). They authors propose that P. monodon should have an important release period before September, in addition to that found by them and other authors in November (Palma, 1994).The larval release period for resident species of the OMZ occurs in winter, which contrasts to that for coastal species (Figure 5). Taking into account that temporal and spatial influences are important to the success of larval release and subsequent recruitment (Cury and Roy, 1989; Stenevik et al., 2003), it is expected that the species present distinct patterns of larval release in contrasting ecosystems (i.e. coastal v/s benthic), since the local forcings that modulate these patterns also are different. It has been suggested that in upwelling areas larval release occurs during periods of increased primary productivity, and/or during periods of reduced risk of offshore advection (Bakun, 1996). In Chile the main period of hatching occurred during the month of September, just as primary productivity in the surface starts to increase (Montecino and Lange, 2009). The abundance and distribution patterns of larvae from three intertidal coastal species were consistent with the expected results for coastal species (Anger, 2001).These observations are both consistent with the peak period for egg carrying in E. analoga (Contreras et al., 1999), as well as with the distribution and abundance of larvae in E. analoga in the Coquimbo region (Flores and Mujica, 2009), and also observed in B. spinimana in Concepción (Yannicelli et al., 2006a). Contrarily, larval release of P. monodon occurs mainly in winter, when Chl˗a levels are at a minimum (Figure 2 and 5) but bottom oxygen levels and temperature rise. The rate of embryo survival and larval hatching success of Nephrops norvegicus, a benthic decapod that inhabits low oxygen environments, decreased when brooding females were maintained in chronic hypoxic conditions (Eriksson et al., 2006), so overall reproductive output success was linked with the environmental oxygen conditions experience during the maternal-caring stage. Guzmán et al. (2016) showed for .P monodon that winter eggs energy content was high since larvae would be released during late winter, a period of poor feeding conditions for larvae, in contrast with late summer broods. Therefore, the time lag between larval release of OMZ resident organisms and the later increase in primary production, as well as the migration of ovigerous females to shallow more

oxygenated area scan be understood if decreases in oxygen can compromise the viability of the embryos and eggs are reach in energetic reserves so larvae could endure low food concentration. Favorable pre-hatching conditions could finally enhance the overall success of a reproductive event. The main period of larval release for N. uncinata, the only subtidal species, occurs in August. This follows the same yearly pattern as P. gaudichaudii. Considering this, it has been described that P. monodon, N. uncinata and Libidoclaea granaria (the latter also an OMZ resident) show similar patterns of temporal and spatial distribution, and vertical migration, releasing their larvae before or at the start of the upwelling season at 37°S (Yannicelli et al., 2006a;Yannicelli et al., 2006b). P. monodon and L. granaria are resident in the OMZ, whereas N. uncinata is an infaunal crustacean that inhabits locations with low oxygen conditions. Therefore, we note that the release period does not fit the classic functional hypothesis (Olive, 1995), and that periods of larval release are not in synchrony with favorable periods for food availability (austral summer) which provide conditions to improve the chances of larval survival and development (Anger, 2001; Guzmán et al., 2016). We propose that successful embryonic

development is key for reproductive success, so improved pre-hatching conditions would favor later larval success, and overall reproductive outcome in a variable environment. 4.3.3 Juveniles The juveniles P. monodon were collected from the benthos during the same time periods in autumn (from the beginning of March to June) and in spring (October to December), and partially correspond to different cohorts. This indicates that the autumn peak (of smaller juveniles) originates from larvae released during spring, while the spring peak of juveniles originates from larvae released during winter. Two recruitment peaks had previously been described by Acuña et al., (2004) for the area. On the contrary, a single peaks (annual cohort) of recruitment had previously been described on the Concepcion coast for P. monodon (Gallardo et al., 1994). It has been suggested that about 36-37°S, recruitment of juveniles to the benthos is a response to optimal benthic conditions, i.e. an increase in oxygen during the autumn months (Gallardo et al., 1994). However, our results show that juveniles are not restricted by benthic oxygen levels (unlike ovigerous females) (Figure 4), and furthermore, the peaks of juveniles benthic abundance occurred during contrasting periods of background oxygen levels (Figure 2 and 6). Consequently, it could be argued that conditions in the water column could determine juveniles benthic settlement, since juveniles keep growing (Gallardo et al 1994) while migrating daily from surface waters to the bottom until definite settlement (Yannicelli et al 2012). In autumn, the conditions change from high

to low primary productivity in the water column, so low pelagic productivity during autumn would not justify the metabolic cost of juveniles vertical migration. Differences in settlement timing between the different benthic populations could be due to the fact that chlorophyl-a levels in Concepción are higher than at 30ºS and remain higher during the entire year in spite of seasonality (Carr and Kearns 2003). The low recruitment observed in the spring of 2010 coincided with low larval abundance and low proportion of ovigerous females registered in the winter of 2010, possibly a consequence of La Niña that affected this area until mid-July of the same year (Braum et al., 2010). One effect of La Niña on the ESP continental shelf is that the OMZ shallower and more intense, possibly result in suboptimal conditions for ovigerous females, embryonic development, and finally the success of larvae. 4.4 The reproductive cycle of OMZ residents Our results support the initial hypotheses, where the seasonal variability of OMZ was tuned with the period of egg carrying by females. The retreat of the OMZ from the continental shelf, in winter elevates oxygen concentrations in the bottom layers of the continental shelf coinciding with the onset of the reproductive period. It is usually accepted that in upwelling systems species synchronize their reproductive periods so larval hatching match the most favorable environmental conditions for larval survival: eg. enhanced food availability and moderate turbulence (Cury and Roy 1989), reduced advective loss (Bakun 1996; Anger, 2001; Thiel and Lovrich, 2011). Nevertheless, P. monodon presents an extended reproductive period producing several annual broods, so recently hatched larvae experience contrasting oceanographic conditions (Thiel et al 2012). According to the results presented and literature, egg carrying females between ~30-37 ° S occur mainly during autumn-winter, during the retreat of the OMZ, that would privilege the success of the embryonic development and hatching of larvae with high energy content. Moreover, in spite feeding conditions might not be optimal during larval release period, larval loss through advection should be lower than during the more intense upwelling periods (Yannicelli et al., 2012;Yannicelli and Castro, 2013). On the other hand, the period of maximum egg carrying female incidence coincided with that of OMZ retreats from the studied area, and gravid female depth distribution varied seasonally above a threshold of oxygen concentration (0.5 ml L-1). Therefore, we propose that reproductive seasonality might respond to the following maternal environmental constraints: i) acquisition of energy for reproduction during summer, when primary productivity (and export to the benthic environment) is

maximum; and ii) brood development at depths and seasons of increased benthic oxygen concentration. The pattern of egg carrying during the winter and the release of larvae in late winter to early spring seen in P. monodon has also been observed in other OMZ residents in this latitude: the yellow squat lobster Cervimunida johni(Wolff and Aroca, 1995;Pool et al., 1996;Palma and Arana 1997), the hake Merluccius gayi gayi (Tascherio et al., 1999), the nylon shrimp Heterocarpus reedi (Canales et al., 2016), in addition to that found in Zoea I of Platymera gaudichaudii in this study. However, the coincidence in the reproductive period in different taxa would indicate an influence of one or more environmental factors that would be forcing the reproductive period of the OMZ residents. As in P. monodon, the relationship between the seasonal variation of OMZ and the reproductive period of these commercial species has not been studied. However, we can infer that the variation of oxygen plays an important role in M. gayi, since Tascherio et al. (1999) report that abundance of mature females and spawning have a negative relation with the upwelling index. This could be related to the decrease in oxygen content that occurs over the shelf area when upwelling conditions prevail. In spite large inter-annual recruitment variability has been detected for the species, no direct association with traditional proxies of inter-annual oceanographic variability (eg temperature) have been found. Our results suggest that inter-annual variability on continental shelf oxygen conditions could influence reproductive success and therefore should be further explored together with population time series. Current models considering global climate change scenarios, predict

intensification of hypoxia as well as OMZ horizontal and vertical expansions leading to shallower OMZ upper limits. Changes in oxygen conditions could have an effect on the reproductive success of P. monodon benthic populations, either restricting the viable brooding period or brooding females depth distribution. This could result in decreases in the number of annual broods per female, hatched larvae quality and finally affect recruitment and adult biomass. Our results point to the relevance of considering OMZ variability in the modulation of resident species biological cycles and expands previous observations of other OMZ resident species. A conceptual model proposed for a single species by Yannicelli and Castro (2013), may also be suitable as a general model of reproductive seasonality valid for HCS residents OMZ (Figure7). If the most important species associated with the OMZ share a reproductive pattern, convergence could be a strong selective pressure to synchronize the period of egg carrying with environmental factors (oxygen and food variability). However, to test these hypotheses ecophysiological studies are needed in ovigerous females for OMZ resident species, to determine the metabolic restrictions

on different scenarios of oxygen and temperature, simulating the seasonal variation of the OMZ in the ESP. ACKNOWLEDGEMENTS The authors thank the Innova-Corfo Project 07CN13 IXM-1 50 for providing biological and oceanographic data. Conicyt Becas Chile 21110922 provide funds for MAG. B Yannicelli, M Ramos and J. Sellanes acknowledge support from Fondecyt (project 1140832), supported data analysis and writing as part of objectives. M. Ramos and B. Yannicelli acknowledge support from FONDECYT (project 1140845). M Ramos, B. Yannicelli and J Sellanes acknowledge support from Chilean Millennium Initiative (NC120030).Our gratitude also goes to the team of the Biological Collections Room (SCBUCN) from the Universidad Católica del Norte (Jorge Aviles), the crew of the scientific vessel Stella Maris II crew and also to Innova Corfo Project lab and field assistants: Sergio Fuentes, Eduardo Flores, Matías Pizarro and Ives Melville. REFERENCES Acuña, E., Alarcon, R., Arancibia, H., Cid, L., Cortés, A., Cubillos, L., Haye, P., León, R., Martínez, G., Neira, S., 2004. Evaluación directa de langostino colorado y langostino amarillo entre la II y VIII regiones, año 2004. Informe Final. Proyecto FIP No 2004-11, 405 pp. Andrade, H., 1986. Observaciones bioecológicas sobre invertebrados demersales de la zona central de Chile. La Pesca en Chile 41–56. Anger, K., 2001. The biology of decapod crustacean larvae, Crustacean Issues, Vol 14. AA Balkema, Lisse, The Netherlands. doi:10.1651/0278-0372(2005)025 Anger, K., Thatje, S., Lovrich, G., Calcagno, J., 2003. Larval and early juvenile development of Paralomis granulosa reared at different temperatures: tolerance of cold and food limitation in a lithodid crab from high latitudes. Mar. Ecol. Ser. 253, 243–251. Antezana, T., 2009. Species-specific patterns of diel migration into the Oxygen Minimum Zone by euphausiids in the Humboldt Current Ecosystem. Prog. Oceanogr. 83, 228–236. doi:10.1016/j.pocean.2009.07.039 Apablaza, P., Palma, S., 2006. Efecto de la zona de mínimo oxígeno sobre la migración vertical de zooplancton gelatinoso en la bahía de Mejillones. Investig. Mar. Val paraíso 34, 81–95. Aste, A., Retamal, M., 1984. Desarrollo larval de Callanasia uncinata H. Milne Edwards, 1837 (decapoda, Callanassidae) bajo condiciones de laboratorio. Gayana (Concepción) 48, 41–56. Baba, K., Ahyong, S.T., Macpherson, E., 2011. Morphology of marine squat lobsters, in: Poore, G., Ahyong, S.T., Taylor, J. (Eds.), The Biology of Squat Lobsters. Csiro Publishing, pp. 1–37.

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Figure 1.Study area Bay of Tongoy (Chile). a) Geographical location; b) spatial distribution of 18 sampling station visited throughout SIPO oceanographic cruises. Symbols: black filled squares: zooplankton sampling+CTD-O; black filled dots: benthic trawling+CTD-O; black filled triangles: benthic trawling and zooplankton sampling + CTD; open circles: only CTD stations. Oxygen was sampled in uneven numbered stations.

Figure 2.Temporal variability of temperature, dissolved oxygen concentration and chlorophyll a concentration at different depths throughout the study period. a and b) Sections of dissolved oxygen concentration (mlL-1) from Station 1 (offshore) to station 9 (inner bay) for Summer (a) and autumn (b); c) Oxygen concentration (mL L-1) at 50 y 500 m. d) Oxygen concentration(mL L-1) at 100, 150 y 250 m. e) Temperature (°C) at 50, 100, 150, 250 m. f) Chlorophyll-a concentration (mg m-3) at 0, 5 y 15 m.

Figure 3.Temporal variability of abundance and depth distribution of ovigerous and non-ovigerous Pleuroncodes monodon Females. a) Ovigerous females abundance as individuals m-2 (grey triangles) and percentage of ovigerous females out of the total sampled females (black filled circles). b) Depth distribution of ovigerous females throughout the sampling period. The positive depths of occurrence of ovigerous females for each sampling date are shown by circles. Circles completely filled in black indicate that 100% of ovigerous females occurred at a single depth that date. Whenever ovigerous females were distributed in two or more depths, black sectors in the circles represent the percentage of them found at each depth. c) Depth distribution of non-ovigerous females. Circles, circles filled in grey and grey sectors in circles as in “b” but for non- ovigerous females.

Figure 4. Cumulative frequency of ovigerous females (OF), non-ovigerous females (NOF), males (M) and juveniles (J) found at different oxygen concentrations throughout the studied period.

Figure 5. Temporal variability of larval stages abundance (ind. 100m-3) for resident (Pleuroncodes monodon and Platymera gaudichaudii), coastal (Emerita analoga, Lepidopa chilensis, Blepharipoda spinimana) and subtidal (Neotryapea uncinata) crustacean species.

Figure 6.Temporal variability of abundance and depth distribution of Pleuroncodes monodon juveniles. a) Temporal variability of abundance (individuals m-2) of Pleuroncodes monodon juveniles (black circles). b) Depth distribution of ovigerous females throughout the sampling period. The positive depths of occurrence of juveniles for each sampling date are shown by circles. Circles completely filled in black indicate that 100% of juveniles occurred at a single depth that date. Whenever juveniles were distributed in two or more depths, black sectors in the circles represent the percentage of them found at each depth.

Figure 7.Conceptual model of the temporal variability of dissolved oxygen concentration and reproductive cycle of Pleuroncodes monodon in Central and South-Central Chile. The climatological annual cycle of oxygen concentration was re-drawn from Yannicelli and Castro (2013), based on monthly oxygen casts from 2005 to 2009 undertaken at the Chilean shelf about 37°S (Fondap COPAS, U. de C.). Chlorophyll-a bar was built based on own results. The main life stages of Pleuroncodes monodon life cycle was drawn from main stream publications, grey literature, SIPO and our own unpublished data. Red dotted lines indicate larval and juveniles vertical migration. Lines and graphs in red indicate the strength and duration of the different developmental and life cycle stages as well as their vertical distribution. Blue lines

indicate relevant periods with respect to OMZ variability and upwelling intensity (upwelling and retreat of the OMZ).

Table 1. Details of data available and analyzed per cruise. Number of total zooplankton stations visited and availability of oceanographic data. Cruise SIPO I02 I03 I05 I06 I07 I08 I09 I10 I11 I12 I13 I14 I15

Date 17/06/2010 03/08/2010 24/11/2010 21/12/2010 20/01/2011 31/03/2011 20/04/2011 27/05/2011 29/06/2011 05/08/2011 01/09/2011 13/10/2011 24/11/2011

Zooplankton (Nº stations) 6 0 9 9 8 9 7 9 7 5 7 9 4

*Measurements CTD and water samples

Oceanographic* yes yes yes NO Yes yes yes yes Yes yes yes yes Yes

Table 2. List and main characteristics of benthic species analyzed. Characteristics include: Latitudinal and bathymetric distribution (latitudes along the west coast of South America); type of habitat: OMZ resident (Z), coastal (C) or subtidal (S); whether species is captured or not by trawl fishery as target species or by catch. Biological characteristics: periods of ocurrence of ovigerous females; incubation time; larval developmental time until megalopa: number of larval stages: n° of zoea stages + M (megalopa),? Non information. Peack Distribut ion

Specie

Bathym etry (Chile)

Habi tat

Taxomom y

Tra wl fishe ries

oviger ous female s

Incuba tion time

Larval Stage

Larval developme nt

40 days (laborat ory, 1113ºC) (Thiel et al. 2012)

5 (+ 3 substage)+ M (Fagetti & Campodonico 1971)

1 month (20ºC) 3 month (11ºC) (Palma 1994)

Pleuroncode s monodon

P m

16ºN37ºS

50-400 m

Z

Anomura: Munididae

target

Jun-Aug (Palma & Arana 1997)

Platymera gaudichaudi i

Pg

38ºN37ºS

40-450 m

Z

Brachyura: Calappidae

bycat ch

?

?

5 incomplete +M (Gallardo & Mujica 2010)

?

Neotrypaea uncinata

N u

0-1 m

S

Axiidae: Callianassi dae

target

?

?

5 + M (Aste & Retamal 1984)

7 a 14 days

C

Anomura: Hippidae

-

Nov-Dec (Contrera s et al. 1999)

29-32 days

5 + 1 (Johnson & Lewis 1942)

3-4 months

C

Anomura: Albuneidae

?

42-62 days (2023ºC)

(Sánchez & Aguilar 1975)

43-61 days (20-23ºC)

(Knight, 1968)

34-52 days (in Blepharipo da occidentali s)

Emerita analoga

Lepidopa chilensis

Blepharipod a spinimana

Mexico to Chile (46ºS).

Ea

58ºN55ºS

Lc

3º 30`S39ºS

Bs

17ºS-39ºS

0-3 m

0-2 m

0-2 m?

C

Anomura: Blepharipo didae

-

-

?

?

Table 3. Results of the GLM (best reduced model) for the influence of environmental condition (oxygen, depth, temperature, chlorophyll-a (at 5 m)) on the presence/absence of ovigerous females (a), non-ovigerous females (b) and juveniles (c). Significant values (p= 0.05) are in bold. Best Models Ovigerous Females

S.E

Z-value

p-value

Model(AIC: 24.143;Pseudo-R2:0.55,residualdeviance: 18.143; d.f:30) Intercept Oxygen Chlorophyll-a (at 5 m)

Non- OvigerousFemales

Estimate

-3.5510 6.9755 -0.8668

1.6106 2.8986 0.4111

Model(AIC: 24.683;Pseudo-R2:0.59,residual deviance: 16.683; d.f:29)

-2.205 2.407 -2.108

0.0275* 0.0161* 0.0350*

Intercept Chlorophyll-a (at 5 m) Juveniles

-0.3708 -0.2150

0.6307 0.2245

-0.588 -0.957

0.5565 0.3383

-1.571 1.163 2.085

0.1163 0.2447 0.0371*

Model (AIC: 38.414;Pseudo-R2:0.20, residual deviance: 32.414; d.f:30) Intercept Chlorophyll-a (at 5 m) Depth

-2.050 0.228 0.019

1.306 0.196 0.009

Highlights  Only the distribution of P monodon ovigerous females is restricted by low dissolved oxygen levels (0.5 ml L-1).  The incidence of P. monodon ovigerous females as well as their depth of occurrence coincide with the periods of warm and oxygenated bottom waters.  Reproductive seasonality of OMZ resident crustaceans differs from that of shallower species.  OMZ seasonal variability could be acting as a selective pressure to synchronize reproductive periods of OMZ resident species.