Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure

Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure Ana Sofia de Sousa Moreira 2013 Dissertação de Mestrado de Ciências e Tecnologia do Ambiente, Especialização em Tecnologias de Remediação Ambiental This work has been already presented in an international congress as a poster: Moreira AS, Carvalho MG, Moreira C, Daros FA, Queiroga H, Santos PT, Correia AT. 2013. Study of the coastal recruitment of Lipophrys pholis in the Portuguese occidental coast inferred from otolith microstructure analysis. COLACMAR. 27-31 October. Ponta del Este. Uruguay. and accepted to be published in an international paper: Carvalho MG, Moreira AS, Moreira C, Queiroga H, Santos PT, Correia AT. In Press. Validation of otolith daily increments in early juveniles of shanny Lipophrys pholis .Journal of Fish Biology. I Acknowledgements This Master thesis was only possible with the collaboration and contribution, directly or indirectly, from various institutions and people, who I would like to express a few words of thanks and profound appreciation, in particular to: Professor Alberto Teodorico Correia, my supervisor, by the availability manifested to guide this work, for the precious help in defining the object of study, the requirement for method and accuracy, the tireless scientific guidance, the critical review of the text, for comments, explanations, opinions and suggestions, by providing indication of some relevant literature, by timely advice, and by the accessibility, cordiality and friendliness shown. The confidence that has always given to me and the continuous stimulation and interest shown become decisive at certain moments of this thesis. Professor Henrique Queiroga, co-supervisor from the Aveiro University, by the help in some research methodologies and by the valuable information given during the workshops under the LarvalSources project; To my colleagues of the Ecophysiology Lab., Margarida Gama Carvalho and Cláudia Moreira, a special thanks for your friendship, companionship, motivation and help, very important daily factors in achieving the objectives of this thesis. Also a special reference to Felippe Daros, for the friendship we created. Thank you for all the help and sharing good times. Thanks to all colleagues who have, somehow, helped in the realization of my thesis and all the members of the LarvalSources project; At last, I am aware that alone none of this would have been possible. So special thanks to my parents, because they are models of courage, for their unconditional support, encouragement, friendship, patience and total assistance in overcoming obstacles that were emerging along this walking. This work has been partially supported by the FCT research project PTDC/BIABIC/120483/2010. II Abstract Lipophrys pholis is a Blenniidae interdital fish usually found in the Portuguese coastal shores. Information available on the ecology of this species is scarce and is mainly from reproductive and behavioral studies, made in some cases from captivity experiments. Spawning takes place from October/November to May in rocky nests, after which demersal eggs hatch and larvae disperse to the coastal area. Early juveniles (recruits) return within two to three months, apparently to a particular set of rock tide pools, suggesting a homing behavior. In the present study, and to assess the primary increment periodicity in otoliths of early juveniles of L. pholis, 90 individuals, ranging from 16 to 39 mm of total length, were captured in a rocky beach in north of Portugal (Cabo do Mundo) and reared in laboratory. Otolith direct validation procedures using fluorescent dyes (alizarin and tetracycline) showed a daily deposition of the primary increments, suggesting that they are reliable sources of age information for L. pholis. Additionally 91 recruits (TL≤30 mm) were collected using a hand net in April/May 2013 during the low tides periods in three rocky beaches along the occidental Portuguese coast (Cabo do Mundo, Peniche and Vale do Homem). The purpose was to estimate the total and recruitment ages by counting the primary increments and by tracking the settlement mark, respectively. In the lab, total length of individuals has recorded and sagittal otoliths were extracted. The relationship between fish length and otolith size (radius and diameter) was explored. After adequate preparation, otoliths were observed in light microscopy. Age ate recruitment ranged between 57 and 73 days, showing a latitudinal pattern of temporal recruitment probably related with the seawater temperature. Future works using microstructural and microchemistry analysis from otoliths of individuals collected in different life stages (e.g. embryos, recruits and adults) can be useful to assess connectivity between the spawning and recruitment areas in L. pholis. Key words: blennies, life history, connectivity, sagittae, daily rings III Resumo Lipophrys pholis é um peixe interdital normalmente encontrado nas zonas costeiras portuguesas e que pertence à família Blenniidae. A informação disponível sobre a ecologia desta espécie é escassa, e resulta principalmente de estudos de reprodução e comportamentais, realizados, em alguns casos, a partir de experiências em cativeiro. A postura ocorre de Outubro/Novembro a Maio em ninhos construídos em praias rochosas, mas depois da eclosão dos ovos demersais, as larvas são dispersas através das correntes pela zona costeira. Os primeiros juvenis (recrutas) regressam dentro de dois a três meses, aparentemente para um conjunto particular de poças rochosas, o que sugere para a espécie um comportamento típico do “homing”. Neste estudo 90 recrutas, entre 16 e 39 mm de comprimento total, foram capturados numa praia rochosa do norte de Portugal (Cabo do Mundo). Para avaliar a periodicidade de deposição dos incrementos primários nos otólitos recorreu-se a marcadores fluorescentes (alizarina e tetraciclina) que demostraram uma deposição diária dos incrementos primários nos otólitos, sugerindo que os otólitos podem ser utilizados com sucesso para estimar a idade de L. pholis. Adicionalmente, foram capturados, com recurso a um camaroeiro, 91 recrutas (TL ≤ 30 mm), em Abril / Maio de 2013, durante as marés baixas em três praias rochosas ao longo da costa ocidental Portuguesa (Cabo do Mundo, Peniche e Vale do Homem). Estimou-se a idade total e a idade de recrutamento contando os incrementos primários e tendo em linha de conta o posicionamento da marca de assentamento, respetivamente. No laboratório, foi medido o comprimento total dos indivíduos e foram extraídos os otólitos. A relação entre o tamanho dos indivíduos e dos otólitos (raio e diâmetro) foi analisada. Após preparação, os sagittae foram observados com um microscópio de luz. A idade no recrutamento costeiro variou entre 57 e 73 dias, sugerindo um padrão latitudinal de recrutamento temporal, provavelmente relacionado com a temperatura da água do mar. A análise micro-estrutural e microquímica futura de otólitos de indivíduos capturados em fases de vida diferente (por exemplo, embriões, recrutas e adultos) poderá ser utilizada com sucesso no futuro para avaliar a conectividade entre as zonas de postura e as áreas de recrutamento em de L. pholis. Palavras-chave: blenídeos, ciclo de vida, conetividade, sagittae, anéis diários IV Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 1 - Índex 1. Introduction ........................................................................................................ - 4 - 1.1. Present knowledge about the ecology of Lipophrys pholis .......................... - 4 - 1.2. Otolith microstructure and microchemistry as an useful ichthyological tool11 - 1.3. Objectives ................................................................................................. - 14 - 2. Materials and methods ..................................................................................... - 14 - 2.1. Validation of otolith daily increments in early juveniles of shanny Lipophrys pholis.. ................................................................................................................. - 14 - 2.2. Age at costal recruitment inferred from the otolith microstructure of early juveniles of shanny Lipophrys pholis ................................................................... - 16 - 2.3. Statistical analysis .................................................................................... - 18 - 3. Results ............................................................................................................ - 19 - 3.1 Validation of otolith daily increments in early juveniles of shanny Lipophrys pholis ............................................................................................................................ - 19 - 3.2. Age at costal recruitment inferred from the otolith microstructure of early juveniles of shanny Lipophrys pholis ................................................................... - 20 - 4. Discussion ....................................................................................................... - 25 - 5. Conclusion ....................................................................................................... - 31 - References ............................................................................................................. - 32 - Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 2 - Index of images Figure 1 - Distribution map of L. pholis [modified from Zander (1986) and Almada et al., (2001)]. ..................................................................................................................................... - 5 - Figure 2 - Photography of an adult L. pholis.(personal captation) .................................. - 6 - Figure 3 - Portuguese Rocky beaches where recruits were collected (modified from Google Earth) ........................................................................................................................ - 16 - Figure 4 - Frequency of fish sizes captured in Cabo do Mundo, Peniche and Vale do Homem (n=91). ..................................................................................................................... - 17 - Figure 5 – (a) UV light photograph of sagittae from L.pholis (TL= 22.0 mm) treated with tetracycline sacrificed at 10 days. (b) Photograph of the same sample under normal light with tetracycline mark (200x)...................................................................................... - 19 - Figure 6 - (a) UV light photograph of sagittae from L.pholis (TL= 23,0 mm) treated with alizarin sacrificed at 30 days. (b) Photograph of the same sample under normal light with alizarin mark (200x)...................................................................................................... - 19 - Figure 7 - Relationship between number of increments reading of marked otoliths, with alizarin (n=27) and tetracycline (n=38), and the number of days in the experiment. . - 20 - Figure 8 – Relationship between the radius and diameter with the fish length (p<0.05). - 21 - Figure 9 – Linear regression relating individual Length (TL) with age at capture from Cabo do Mundo (n=20), Peniche (n=20) and Vale do Homem (n=17) ........................ - 22 - Figure 10 - Sagital sections of L. pholis otoliths: (a) Type Ia settlement mark; (b) Type Ib settlement mark. ............................................................................................................... - 22 - Figure 11 - Age at coastal recruitment (mean ± SD) in otoliths of L. pholis collected in three sampling locations (Cabo do Mundo n=20, Peniche n=20 and Vale do Homem n=17) along the Portuguese coast. The locations marked with different letters above the error bars are significantly different (Tukey-test, P<0.05) ....................................... - 23 - Figure 12 – Mean Increment width (µm) from primordium to the edge of the otolith increment. The individuals captured were grouped according to the sampling sites and SM was identified (↓). (Cabo do Mundo n=20, Peniche n=20 and Vale do Homem n=17) ...................................................................................................................................... - 24 - Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 3 - Index of tables Table 1Abiotic parameters controlled in seawater aquaria during the otolith validation studies (mean ± standard error). ............................................................................. - 16 - Table 2 – Surface sea water average temperatures (ᵒC) in three points of Portuguese coast in March, April and May 2013 provided by Instituto Hidrográfico da Marinha Portuguesa (nm, means nautical miles). ................................................................. - 17 - Table 3 - Information relative to the total number of captured individuals (n), Total Length in mm (TL: mean ± S.D.) and range (mm). .................................................. - 21 - Abbreviations ICP-MS: Inductively Coupled Plasma Mass Spectrometry LM: Light Microscopy SEM: Scanning Electron Microscopy SM: Settlement Mark TL: Total Length Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 10 - Juveniles recruited in early winter in Portugal, are probably able to reach the minimum size necessary to reproduce in the next season, since they could grow continuously for several months (Faria et al., 1996). Recruitment of fish < 20 mm ceases 3 months after the end of the breeding season. Recruitment is an operational process: that refers to the first age class in a population (0+) which includes the settlement and survival of settled individuals, and is influenced by biological events occurring during the planktonic stage and settlement processes, as well as postsettlement mortality (Keough & Downes, 1982 Faria et al., 1996). Larval availability and post-recruitment processes in rocky intertidal invertebrate populations have a relative importance. It means that in a few cases larval supply may limit population size, but in others, larval supply is more than sufficient to ensure population renewal (Connell, 1985). The predominant mechanism that limits population size is density dependent post-recruitment mortality, which also seems to occur with L. pholis (Connell, 1985). The photoperiod and temperature can affect the synchronism of the annual cycle of gonadal maturation in L. pholis (Shackley & King, 1977). The temperature appears to be an important abiotic trigger stimulus for the onset of reproduction in this species (Almada et al., 1990a). For species with an extended reproductive season, such as L. pholis, it is probably important to limit the duration of the each breeding season in order to restore the nutritional reserves and to allow growth of the adult itself, since fecundity tends to increase with the individual body size (Wootton, 1990). At higher latitudes the embryos remain in a dormancy stage throughout the coldest months, being released in the spring when conditions are favorable for feeding and growth of the larvae (Crisp, 1954). This behavior suggests a principle that may be generally applicable to all marine animals which have an oceanic planktonic larval stage. The breeding cycles are highly regulated in a way that the larvae hatch during the season in which is most favorable for finding planktonic food (Faria et al., 2005). The previously papers provide important information about the shanny concerning its reproductive biology, ecology and behavior. However the knowledge about the population structure, fish movement patterns and connectivity is, at present, scarce. L. pholis could be used as a model in biology to understand connectivity of marine populations, monitor dispersal in planktonic fish larvae and to study fish coastal recruitment processes. Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 11 - 1.2. Otolith microstructure and microchemistry as an useful ichthyological tool Important information for ecological research and fisheries management is obtained from fish otoliths. Otoliths are inner ear stones, made of calcium carbonate (CaCO3) that play an important role in balance and auditory reception of teleost fish (Popper & Platt, 1993). The CaCO3 is crystallized mainly in the mineral form of aragonite (Carlström, 1963) due to the action of an organic matrix (otolin) in which acidic amino acids predominate (Degens et al., 1969). There are three pairs of otoliths (sagittae, lapilli and asterisci) encapsulated within the otic vesicles of the fish head which vary considerably in size, also being formed in different ontogenetic stages. The sagittae and lapilli generally form earlier in development than the asterisci, which in some species do not form until after hatching. The sagittae are most often used otoliths for ageing purposes and other analyses because they are the largest, earliest formed and easiest to extract of the three pairs (Green et al., 2009). Otolith formation starts with a primordium, which is generally the first calcified tissue in the embryo (Dunkelberger et al., 1980). The nucleus is formed when the first discontinuous unit (Dunkelberger et al., 1980) is laid down which corresponds to hatching, first feeding, or start of activity (Brothers & McFarland, 1981; Morales-Nin, 1992), although some species with long embryonic periods may start forming increments before hatching (Morales-Nin, 2000). Otolith microstructure can provide useful information on life history traits of fish allowing to determine pelagic larval durations, to reconstruct settlement patterns and to investigate information provided by annual and daily increments (Choat & Robertson, 2002; Thorrold & Hare, 2002). Age determination of fishes based on periodic growth increments in otoliths has become a routine tool in fisheries science over the last century. Over 1 million fish were likely estimated through otoliths by fisheries scientists around the world (Campana & Thorrold, 2001). The structure of otoliths is particularly useful to reveal ontogenetic or environmental patterns changes experienced by individual fish. Specific variation in the environment can induce a specific mark in the otolith structure which could be useful afterwards as a reference mark to compare individual life history trait responses (Sponaugle & Pinkard, 2004). The biological significance of first mark in otoliths is probably species specific and may correspond to several life history events such as yolk-sac absorption, hatching or first feeding (Wright et al. 2002b). In some species, such as tropical clupeids (e.g. Stolothrissa tanganicae and Limnothrissa miodon), otolith growth may Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 12 - initiate increment deposition at yolk sac absorption up to 2–8 days after hatching (Kimura, 1995). The next major steps for a developing fish can be metamorphosis (i.e., the transition between larval and juvenile life) and then settlement (i.e., final installation in one specific environment), a particularly sensitive stage in tropical areas and especially for reef species (Leis, 1991). The metamorphosis stage varies greatly between species but it is generally a strong transition associated with the end of ossification and for that reason it constitutes a tremendous physiological and morphological stress for the fish. Metamorphosis also can be associated with habitat change including changes in environmental conditions and behavioral changes, for example in mesopelagic marine species (Myctophidae, Photichthyidae) (Wilson & McCormick 1997, 1999; McCormick et al., 2002). Settlement is a transitional event that is frequently evident in otoliths and of interest in ecological studies of juvenile fish (Green et al., 2009). Settlement marks, can be gradual or abrupt, are frequently species-specific, and can vary within species among local populations (Wilson & McCormick, 1997, 1999). Studies on recruitment of fish in the tropics often rely on the measurements of settlement marks. Growth rates of pelagic larvae which have been argued to be one of the principal determinants of recruitment success have also been estimated from otolith’s microstructure (Bergenius et al., 2002). Otoliths grow by successive deposition of increments, the so-called primary increments, which constitute bi-partite structures each composed of one L-zone and one D-zone. The L-zone is a band rich in calcium carbonate crystals, translucent to light (LM) and appearing raised in scanning electron microscopy (SEM), with an increment width varying between 0.4 and 10 μm. The D-zone is a band rich in organic material, opaque to LM and appearing as a groove in SEM, with an increment width smaller than 1 μm (Panfili et al., 2002). The width of a primary increment (also named micro-increment) usually ranges between 1 and 12 μm (Pannella, 1974). Micro-incremental patterns in the otoliths vary from sub-daily to daily, lunar and seasonal scales (Campana & Neilson, 1985). Rhythmical patterns in the deposition of increments in the otoliths of fish are the basis of age estimation and depends of an endogenous rhythm externally calibrate by the photoperiod (Morales-Nin, 2000). The influence of lunar rhythms in the recruitment of coral reef species has been demonstrated since it alters the otolith microstructural pattern (Sponaugle & Pinkard, 2004). Food intake and food deprivation also have an influence on microstructure, width and periodicity of increments in otoliths (McCormick & Molony, 1992; Molony, 1996; Massou et al., 2002). Stress-induced marks indicate the cessation of otolith Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 13 - growth, which is rare phenomenon, and appear under transmitted light microscopy as opaque, regular, thin marks (Pannella, 1980). Salinity or dissolved oxygen concentration are exogenous factors potentially affecting micro-increment periodicity, but they have scarcely been studied. It is known that salinity induces changes in otolith composition at the microstructure level, yet little is known on how salinity can affect microstructure deposition (Green et al., 2009). Oxygen depletion in the water has been hypothesized to cause disruption of microstructure deposition in Tilapia sp. (Pannella, 1980) and even resorption of the otolith edge in Carassius auratus (Mugiya & Uchimura, 1989). The micro-chemical composition analysis has added another dimension to otolith studies (Green et al., 2009). Geochemical signatures encoded within otoliths are natural tags that are continuously recording information throughout the lifetime of a fish and can be used to infer differences in a range of ecological characteristics of a species such as migration history (Radtke et al., 1996; Tzeng & Tsai, 1994), temperature history of fishes (Radtke, 1989; Radtke et al., 1990; Townsend et al., 1995) and differentiate among fish stocks (Campana et al. 1994; Milton et al., 1997; Thorrold et al., 1998b). Otolith chemistry has been used to validate microstructural features in otoliths such as annual increments (e.g. Kalish, 2001; Andrews et al., 2005), metamorphosis and settlement marks (e.g. Arai et al., 1997; Shen & Tzeng, 2002). Inductively coupled plasma mass spectrometry (ICP-MS) was introduced in 1980 and was originally conceived for solution-based analysis but laser ablation devices were quickly developed that could be coupled with ICP-MS instruments to provide in situ analyses of otoliths. The ability to analyze multiple elements in otoliths at concentrations down to ultra-trace levels has led to new insights into larval dispersal pathways in both tropical and temperate oceans (e.g. Swearer et al., 1999; FitzGerald et al., 2004). New techniques have been developed that use the otolith as a storage device for unique transgenerational isotope labels rather than as a record of natural environmental variations (Thorrold et al., 2006). Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 14 - 1.3. Objectives The main purpose of this work will use the microstructure of otoliths to improve the knowledge about some life history traits of L. pholis. Firstly, the primary increments deposition growth rate will be validated through the use of fluorescent markers (alizarin and tetracycline) in controlled-laboratory experiments; Secondly, the relationship between fish length and otolith size (radius and diameter) or total age will be explored; Thirdly, by counting the primary increments and by tracking the settlement mark on sagittae, the age of individuals at coastal recruitment will be estimated; and Fourthly, the relationship between the available record of the coastal Portuguese sea-water temperatures and the age at coastal recruitment in L. pholis will be assessed. 2. Materials and methods 2.1. Validation of otolith daily increments in early juveniles of shanny Lipophrys pholis Fish Sampling and Acclimation The fish sampling took place in Cabo do Mundo (41º13’N, 8º42’W) in March and August 2013. A total of 90 early juveniles were collected using a small hand net in the pool-tides during the low tide periods. The individuals were transported in refrigerated box with continuous oxygenation to the laboratory and kept in quarantine for two weeks to use in two independent experiments. Experimental Design Forty five recruits (TL of 19.9 ± 2.3 mm; range 17-23 mm) were placed in a 25 liters aerated seawater aquarium, followed by a buffered bath containing alizarin red S (Sigma-Aldrich, A5533, 100 mg/L) and another 45 individuals (TL of 26.1 ± 5.3 mm; range 16-39 mm) in tetracycline hydrochloride (Sigma-Aldrich, T3383, 400 mg/L) for 24 hours. The aquarium was kept in the dark to prevent light-degradation of the fluorescent chemical and the fishes were unfed and undisturbed. Bath immersions were buffered by adding potassium hydroxide (KOH, ≥85%) solution to adjust pH to Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 15 - 8.41 and 8.02, respectively. After 24 hours, the fish were removed and placed in three different seawater aquaria (replicates) with about 25 L of seawater, fed daily “ad libitum” with frozen shrimp and fresh mussels and with a controlled photoperiod of 12D:12N. During the experiments all tanks were monitored daily for water temperature, pH and salinity (Table 1), using a multi parameter probe (YSI, 556 MPS). Fish were sacrificed following 10, 20 and 30 days of exposure. Otoliths from individuals that died before the end of the experiments were not used. Otolith Preparation and Observation Both sagittal otoliths were carefully extracted with plastic forceps under a magnification of 15x binocular stereo microscope (Meiji, EMZ-13TR) and cleaned with 70% ethanol to remove all organic tissues. Left otoliths were mounted on microscope glass slides with the convex side up using a drop of epoxy resin (Buehler, EpoThin). Resin was allowed to dry at room temperature during 24 hours. Otoliths were grounded with silicon carbide paper (Hermes, 2500), wetted with Milli-Q-Water and moving circularly the glass slides. During this procedure frequent checks were made in light microscope (Olympus, CX41), using a clearing mixture (ethanol:glycerol, 1:1), to see if micro-increments were most visible until the core was revealed. At the end, otoliths were polished with alumina paste (Struers, AP-Paste). The detection of the fluorescent band was carried out by viewing the otolith through a compound microscope (Leica DM6000B) with UV light resource at 200x and 1000x, and thereafter viewed again under light microscopy to enumerate the number of growth increments between the fluorescent mark and the otolith edge (Fig. 6). Microphotographs were taken using a USB digital camera (Olympus, SC 30) and otolith morphometric measurements were made using a free software program (Olympus, MeasureIT SC30).The UV filters used were Leica filter N2.1 for alizarin red and Leica filter D for tetracycline. The number of increments was blind counted by three independent readers. The coefficient of variation was lower than 10%. The otolith growth rate has also estimated by measuring the maximum radius between the fluorescent mark and the otolith edge and dividing by the time elapsed. Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 16 - Table 1Abiotic parameters controlled in seawater aquaria during the otolith validation studies (mean ± standard error). Abiotic Parameters Replicates Temperature (ºC) Salinity (psu) pH Alizarin 1 16.2±0.5 35.3±0.6 7.96±0.06 2 16.0±0.5 35.3±0.6 7.92±0.05 3 15.8±0.5 35.4±0.6 7.98±0.06 Tetracycline 1 16.1±0.2 35.0±0.6 8.74±0.06 2 15.7±0.2 35.0±0.6 8.75±0.05 3 15.1±0.2 35.0±0.6 8.77±0.04 2.2. Age at costal recruitment inferred from the otolith microstructure of early juveniles of shanny Lipophrys pholis Biological Sampling Three sampling campaigns were conducted in April/ May (2013) and a total of 91 young juveniles fishes were collected in three rocky beaches equally spaced along the Portuguese coast from North to South (Cabo do Mundo: 41º13’N, 8º42’W, Peniche :39º26’N, 9º13’W, and Vale do Homem : 37º22’N, 8º49’W) (Fig. 3). Since temperature seems to influence the growth of the species, the sea surface water temperatures in these regions were retrospectively considered for 3 months (average mean age of the individual captured). These data were available from the Instituto Hidrográfico da Marinha Portuguesa (Table 2). The specimens smaller than 30 mm (recruits) were collected with hand-nets in rocky pools during the low-tide period. The recruits were kept in sea-water, placed in ice and rapidly transported to the laboratory. All fishes were measured (total length: TL, mm) and distributed by size classes at Figure 3 - Portuguese Rocky beaches where recruits were collected (modified from Google Earth) Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 17 - intervals of 1mm (Fig. 4). The individuals from each size class were frozen (-20ºC) together in Eppendorf tubes filled with seawater. Table 2 – Surface sea water average temperatures (ᵒC) in three points of Portuguese coast in March, April and May 2013 provided by Instituto Hidrográfico da Marinha Portuguesa (nm, means nautical miles). Sea Surface Temperatures (ᵒC) Buoys position Coastline distance (nm) March April May Mean Leixões 11 12.7 13.6 13.0 13.1 Nazaré 4 13.2 14.0 14.0 13.7 Sines 3 14.5 15.5 15.5 15.2 Figure 4 - Frequency of fish sizes captured in Cabo do Mundo, Peniche and Vale do Homem (n=91). Otolith Preparation and Observation Otoliths were carefully extracted and cleaned with Mili-Q-Water. Left otoliths were mounted on microscope glass slides with the convex side up using a drop of epoxy resin (Buehler, EpoThin). Whole otoliths were photographed in a light microscope (Olympus, CX41) coupled to an USB digital camera (Olympus, SC30) at 200x and 400x magnification. The radius, diameter and increment width of sagittae were measured (µm) in their longest axis using a free software program (Olympus, MeasureIT SC30). After the measurements, the otoliths were manually grounded in the sagittal plane with silicon carbide paper (Hermes, 2500) and polished with alumina paste (Struers, AP Paste). 0 2 4 6 8 10 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Frequency TL (mm) Cabo do Mundo Peniche Vale do Homem Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 18 - Final images were worked using a computer program (Olympus, AnalySIS getIT SC30). If needed, successive series of microphotographs from each otolith were made to obtain a complete image of the otolith radius. To count the micro-increments the quality of the otolith’s images were sharpen using a free software (GIMP2.8.4).To determine the total age of recruits at the moment of capture, 16 days were addicted to the counted primary increments (Faria et al., 2002), which corresponds to the number of days before hatching (embryonic period). It was assumed that the first daily ring should represent the hatching check. The number of daily increments was blind counted by three independents readers. Only otoliths in which variation coefficient were lower than 10% were used (average counts). Settlement marks in the otolith were visually identified by the same reader using the optical density transitions and the abrupt change in increment widths (McCormick, 1994). 2.3. Statistical analysis Statistical analyses were performed using SigmaPlot 11. All analyses were carried out according to the common statistical procedures (Zar, 1996). Analysis of covariance (ANCOVA) was used to compare replicates (linear regressions slopes), in alizarin and tetracycline experiments. Since there was no significant differences in replicates (P>0.05), only one linear regression for each treatment was used for data analysis. To assess the daily growth increment periodicity, the slopes of the linear regressions were tested through one sample t-test (H0=1). Linear regression between otolith measurements (radius and diameter) and total length of recruits captured at Cabo do Mundo, Peniche and Vale do Homem were analyzed. The total length of recruits among locations was tested using one-way analysis of variance (ANOVA), followed by a Tukey post hoc test. Regression analysis was conducted on the recruits length and age at capture. ANOVA was used to explore the individual age at recruitment between locations, also followed by a Tukey post hoc test. A level of significance () of 0.05 was used. Data are presented as mean values ± standard deviation (S.D.). Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 19 - 3. Results 3.1 Validation of otolith daily increments in early juveniles of shanny Lipophrys pholis Alizarin and tetracycline appeared as distinct bright red and yellow rings, respectively, when viewed under ultraviolet light in the otoliths of all early juveniles of L. pholis [Figs. 5a and 6a). Both marks were also clearly visible when viewed with normal light microscopy (Figs. 5b and 6b). Figure 5 – (a) UV light photograph of sagittae from L.pholis (TL= 22.0 mm) treated with tetracycline sacrificed at 10 days. (b) Photograph of the same sample under normal light with tetracycline mark (200x) Figure 6 - (a) UV light photograph of sagittae from L.pholis (TL= 23.0 mm) treated with alizarin sacrificed at 30 days. (b) Photograph of the same sample under normal light with alizarin mark (200x) No significant differences were detected among regression slopes (i.e. replicates) within each experiment (ANCOVAs: d.f. = 2.21, P > 0.05 and d.f. = 2.32, P > 0.05, for alizarin and tetracycline, respectively). The regression of number of (a) (b) (a) (b) Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 26 - size of individuals and life stage of fish, more than to the high concentration of dyes and the duration of the immersion period (Hettler, 1984; Vigliola, 1997; Liu et al., 2009). Size of fish could be an explanation of the high mortality of alizarin in this study. Fish used for alizarin (19.9±2.3 mm) were smaller than that used for TC (26.1±5.3 mm) and are probably more vulnerable to the marking and handling procedure. However mortality rates of 35% in successful treatments were considered tolerable for hatchery mass-marking programmes (Eckmann, 2003). Our study also showed a significantly very good relationship between the number of micro-increments in otoliths and the true age of individuals during the experimental marking period. Furthermore, the obtained slope of the linear regressions was very close to 1. These data clearly indicate that the primary increments in sagittae were deposited in a daily basis in L. pholis. No reported validation studies exists on otoliths growth increment periodicity for species belonging to Blennidae. However, Related families, like small cryptobenthic fishes, belongs to gobbiidae family have been documented to present a daily increment formation in otoliths (Hernaman et al., 2000). Our data provides, for the first time, evidence that daily growth increments are reliable sources of age information for L. pholis. This information means that the otolith microstructure of L. pholis can be useful to understand the recruitment process and the connectivity and dispersal movements of the species. Results from this study also indicate that under artificial conditions the daily growth rate of the otoliths for early juveniles of L. pholis was 1.25 ± 0.30 µm. Similar values was obtained for juveniles of and Diplodus putazzo, with a mean growth rate of 1.9 ± 0.5 µm/day and the same value for Diplodus vulgaris with 1.9 ± 0.5 µm/day (Villanueva & Moli, 1997). For Odontesthes bonariensis, the mean growth rate was 3.3 ± 1.2 µm/day (Brown & Fuentes, 2005) and for juveniles of Limnothrissa miodon, ranged from 1.6 to 3.4 µm/day (Meisfjord, 2006). Microstructure analysis of L. pholis otoliths represents an efficient tool for the analysis of life history traits, and provides a range of information that had not yet been obtained for this specie. The age, size and body composition of a newly metamorphosed fish represents the product of all planktonic influences experienced, such as temperature, feeding history and parentage (Green et al., 2009). The easiest way of overcoming differences in pelagic life histories experienced by larvae when attempting species comparisons is by collecting samples over a wide range of environmental conditions. This will be Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 27 - particularly important for fishes with long larval durations and high associated variability (Victor, 1986). The otolith measurements (radius and diameter) in this study showed a positive relationship between fish length. As expected, and described by other authors, fish size and otolith length are related (Alhossaini & Pitcher, 1988; Hovenkamp, 1990; Hovenkamp & Witte, 1991). Because of their function in maintaining the balance of the fish, otoliths tend to grow as the fish grows (Campana, 2004). Many marine organisms, including cryptobenthic fishes, have complex life cycles divided in two main phases: i) the larval stage, which is generally spent in the plankton as gametes and larvae, ii) and the juvenile and adult phases, which starts when the larvae settles into benthic habitats (Roughgarden et al., 1988). Between these phases there is a transition period (i.e. settlement), which is characterized by more or less abrupt morphological and physiological changes (Leis, 1991). Specific variation in the environment can induce a specific mark in the otolith structure which could be used as a reference mark to check some fish life history traits (Sponaugle & Pinkard, 2004). Settlement is one transitional event that is frequently evident in the otoliths and of interest in ecological studies of juvenile fish and can be recorded in very different ways (Green et al., 2009). Otolith settlement marks in reef species can be grouped into categories based on changes in increment width and optical qualities of the mark (Wilson & McCormick, 1999). Fishes with abrupt changes in otolith increment width associated with settlement (Type I) were found to settle during a period of a decrease otolith growth, a few days after reaching their peak growth. The peak in otolith growth may represent the time at which the otolith can function efficiently to meet a species' sensory requirements while in the pelagic realm (Wilson & McCormick, 1999). In our study, two settlement mark types were identified, namely Ia and Ib, both characterized by a sharp decrease in increment width across the settlement mark completed within a few increments. These different types of settlement marks observed in L. pholis corresponds to a single transition increment (type Ia) or a few transition increments (type Ib) (Wilson & McCormick, 1999). Beldade et al. (2007) investigate 10 temperate cryptobenthic species belonging to three families: Gobiidae, Gobiesocidae and Blenniidae. Two subtypes of settlement marks (type Ia and Ib) were found among individuals of the same species. A similar scenario was also observed for some species (e.g. Acanthurus olivaceus, Pomacentrus amboinensis, P. wardi) which increment width decreases as much as 50% around the settlement-mark (Wilson & McCormick, 1999). In total, 80% Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 28 - of the species examined for Wilson & McCormick (1999) displayed this dramatic reduction in increment width over settlement (characteristic of Type I). Fundamental knowledge of dispersal and connectivity of aquatic organisms can be useful to understanding the biological and hydrodynamic processes involved in the transport of larvae and deriving larval origins. Natal origins and destination points provide the basic data in connectivity studies (Cowen, 2007). Time scales of larval development and behavioral capabilities, including vertical migration, play an important role in the evolution of life history strategy of individuals (Sponaugle et al., 2002; Cowen, 2002). Population connectivity of benthic marine organisms occurs primarily during the pelagic larval stage when individuals either return to their natal location to settle, or disperse and settle some distance away from their natal population (Cowen, 2007). Reconstructing settlement patterns and recruitment pulses can be used to estimate persistence of cohorts in populations and infer the relative importance of recruitment processes to adult’s population sizes and dynamics (Victor, 1983). The number of daily increments before the settlement mark indicates an age at recruitment of 73±7 days, 62±6 days and 57±6 days in north, center and south, respectively. Our results clearly reveal a latitudinal pattern of coastal recruitment, probably related with the seawater temperature. The total age for recruits ranged between 61 and 125 days. McCormick (1994) examined the variation in the size of Upeneus tragula at settlement over 8 samples from single station off Lizard Island during November and December 1991. Fishes that settled early in the season were found to be significantly smaller than fishes that settled later (November: 26.9 mm; December: 28.1 mm). Furthermore, the mean age at recruitment did not differ between months (32.2 and 31.4 days respectively). The Gobiidae studied by Beldade et al. (2007) presented a mean pelagic larval duration (DPL) ranging from 19 to 30 days. The only Blenniidae captured in the study, P. pilicornis, had the longest DPL among the species analyzed (33 days). The effect of temperature on the growth of the otolith has been intensively studied and generally shows a positive relationship between otolith growth and temperature (Campana & Neilson, 1985). The possible roles of photoperiod, air and sea temperatures in the control of reproduction in L.pholis were investigated (Shackley & King, 1977). These authors concluded that both photoperiod and temperature seem to affect the course of the annual cycle of gonadal maturation. More recently it has been suggested that the temperature is a more effective factor than photoperiod in triggering the onset of reproduction in this species (Almada et al., 1990a). Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 29 - The relationship between otolith growth and environmental factors was evaluated by comparing age at recruitment of L. pholis and the seawater temperature between sampling sites. As has been done for pomacanthids (Thresher & Brothers, 1985), labrids (Victor, 1986) and pomacentrids (Wellington & Victor, 1989), temperatures experienced by larvae should be reported, to enable comparisons of larval duration among geographic regions. The sea surface water temperature seems to influence the growth of the species. In this study the water temperature was considered for coastline distances between 3 nm and 11 nm, but in the tide pools this temperature can have abrupt daily and or sazonal changes. The temperature in our study showed a variation (Vale do Homem > Peniche > Cabo do Mundo: 15.2 ° C > 13.7 ° C > 13.1 ° C) in the period between March and May 2013. In regions with higher seawater temperatures young juveniles recruit earlier to the coasts. For instance, one individual of L.pholis with a TL of 21.0 mm in the north, centre and south have different ages of recruitment. It means that in the northern fishes has a slow growth comparatively with the most southern individuals, meaning that they settled later. We can conclude that the temperature can influence the fish growth. The temperature is considered an important factor in the early life stages of fishes and has influence in recruitment (Hovenkamp & Witte, 1991). For Hovenkamp & Witte (1991) settlement age is lower at higher temperatures, indicating that development appears to be more related with temperature conditions than with size of the individuals in settlement. McCormick & Molony (1995) suggested that progeny spawned early the extended reproductive season and developing cooler water are likely to have slower growth rates, but may metamorphose and settle at an older age and larger size, than those spawned into warmer waters later in the season. This prediction is supported by latitudinal and regional differences in size or age at settlement suggested in other studies (Randall, 1961, Thorrold & Milicich, 1990). Geffen et al. (2011) describes higher growth rates for juveniles of Pleuronectes platessa at higher temperatures, and according Alhossaini et al. (1989), the size reached in nurseries after the first growing period is also critical for recruitment. Relative daily growth can be obtained by examining the width between successive increments. The comparison of increment widths during a particular stage of life among individuals provides a relative measure of somatic growth (Green et al., 2009). Increment widths measured in L. pholis otoliths showed variations between locations, there is an increase in width from north to south (Cabo do Mundo: 2.18±0.55 Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 30 - µm/day; Peniche: 2.29±0.56 µm/day; Vale do Homem: 2.90±0.62 µm/day). It means that fishes with more age (north) have thinner increments than younger fishes (south). The patterns of daily growth of otoliths during early development, in relation to environmental factors, have been studied by time-series analysis of increment width data (Maillet & Checkley, 1991; May & Jenkins, 1992; Ralston, 1995). Observations in the field and in the laboratory have shown that micro-increment width may change in response to temperature and diet, although the period of microincrement deposition remained daily (Gauldie & Radtke, 1990). Food privation may result in smaller increment widths, but not in the cessation of microincrement deposition (Massou et al., 2002) since the fish has enough body energy reserves (Campana & Neilson, 1985). Differences between increment widths before and after the SM were clear for L. pholis. Age at recruitment estimated by counting daily increments in otolith match with this differences in increment widths. Sagittae increment widths measured in this study are consistent with the increment widths reported for other fish families (Wilson & McCormick, 1999). Studies on recruitment of fish in the tropics often rely on the measurements of settlement marks. Growth rates of pelagic larvae which have been argued to be one of the principal determinants of recruitment success have also been estimated from otoliths (Bergenius et al., 2002). The identification of settlement location is also possible using the differences in individual growth rates (Danilowicz, 1997a). Future works using microstructural and microchemistry analysis from otoliths of individuals collected in different life stage (e.g. embryos, recruits and adults) can be used to assess the movement patterns and habitat connectivity in L. pholis. Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 31 - 5. Conclusion This thesis have achieved to establish a daily deposition of the primary increments on sagittae of L. pholis suggesting that otoliths can be used to ageing purposes in this species. This information was used to estimate the age of L. pholis recruits collected along the Portuguese coast. Furthermore two types of settlement marks were identified by light microscopy. Larval stage duration appears to be highly dependent of seawater temperatures, but size appears to be a key process to trigger the settlement process. Reconstructing early life history patterns and recruitment pulses can be used to estimate persistence of cohorts in populations and infer the relative importance of recruitment processes to adult’s population sizes and dynamics. Future studies should also use otolith microchemistry analysis to infer about the population structure and coastal connectivity of shanny. Faculdade de Ciências da Universidade do Porto Life history of shanny Lipophrys pholis in the Portuguese occidental coast: new insights from otolith microstructure - 32 - References Alhossaini M & Pitcher TJ (1988) The relation between daily rings, body growth and environmental factors in plaice, Pleuronectes platessa L., juvenile otoliths. Journal of Fish Biology 88: 409-418. Alhossaini M, Liu Q, Pitcher TJ (1989) Otolith microestructure indicating growth and mortality among plaice, Pleuronectes platessa L., post-larval sub-cohorts. Journal of Fish Biology 35: 81-90. 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