SCIENTIA MARINA Scientia Marina Sci Mar 0214-8358 Consejo Superior de Investigaciones Científicas sm5045 10.3989/scimar.05045.03A Articles Evaluation of staining techniques for the observation of growth bands in tropical elasmobranch vertebrae Evaluación de técnicas de tinción para la observación de bandas de crecimiento en vértebras de elasmobranquios tropicales Staining techniques in vertebrae of tropical elasmobranchs https://orcid.org/0000-0002-5515-968X Pérez-Rojas José G. jgperez@squalus.org https://orcid.org/0000-0002-4595-3679 Torres-Palacios Katherine ktorres@squalus.org https://orcid.org/0000-0002-2378-5512 Uribe Amalia amauribe17@gmail.com https://orcid.org/0000-0002-6758-7729 Navia Andrés F. anavia@squalus.org https://orcid.org/0000-0003-2220-6969 Mejía-Falla Paola A. pmejia@squalus.org Fundación colombiana para la investigación y conservación de tiburones y rayas, SQUALUS. Calle 10A No. 72-35, Cali, Colombia. Centro Interdisciplinario de Ciencias Marinas, Instituto Politécnico Nacional, Avenida Instituto Politécnico Nacional s/n, Colonia Playa Palo de Santa Rita, Apartado Postal 592, 23096 La Paz, Baja California Sur, México. Grupo Biología CES, Programa Biología, Universidad CES. Calle 10A # 22 – 04, Medellín, Colombia. Grupo de Investigación en Ecología, Universidad del Valle, Departamento de Biología, Sección de Zoología, A.A. 25360. Cali, Colombia. Massutí E. Editor 31 12 2020 2020 84 4 343 354 10.3989/scimar.05045.03A 24 02 2020 26 06 2020 08 09 2020 © 2020 CSIC 2020 This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License. SUMMARY

The aim of this study was to assess the suitability of different vertebrae staining techniques for the visualization and counting of growth bands in tropical species of batoids (Narcine leoparda, Urotrygon aspidura, Hypanus longus, Potamotrygon magdalenae) and sharks (Alopias pelagicus, Carcharhinus falciformis, Sphyrna lewini, Sphyrna corona and Mustelus lunulatus). Different cutting thicknesses and staining protocols were tested, analysing the precision and bias of each combination to identify the most accurate technique for estimating age. Vertebral sections of 0.4 mm were more suitable for batoids, except for Narcine leoparda; for this species and for all the shark species assessed, sections of 0.5 mm are recommended. Different combinations of stain and exposure time were required to achieve the best visualizations of vertebral growth band pair for the shark and ray species. Intraspecific variation occurred among vertebrae size of batoids. Our results confirm the importance of defining a suitable species-specific protocol for sectioning and staining hard structures before carrying out an age and growth study to improve the reliability of the age estimates.

RESUMEN

El objetivo de este estudio fue evaluar la efectividad de diferentes técnicas de tinción de vértebras en la visualización y el conteo de bandas de crecimiento en especies tropicales de batoideos (Narcine leoparda, Urotrygon aspidura, Hypanus longus, Potamotrygon magdalenae) y tiburones (Alopias pelagicus, Carcharhinus falciformis, Sphyrna lewini, Sphyrna corona y Mustelus lunulatus). Se probaron diferentes espesores de corte y protocolos de tinción, analizando la precisión y el sesgo de cada combinación para identificar la técnica más precisa para estimar la edad. Las secciones vertebrales de 0,4 mm fueron más adecuadas para batoideos, excepto para Narcine leoparda; para esta especie y para todas las especies de tiburones evaluadas, se recomiendan secciones de 0,5 mm. Se identificaron diferentes combinaciones de tinción y tiempo de exposición para lograr las mejores visualizaciones de las bandas de crecimiento vertebral en las especies de tiburones y rayas. En los batoideos se identificó variación intraespecífica de acuerdo con el tamaño de las vértebras. Nuestros resultados confirman la importancia de definir un protocolo especie-específico adecuado para cortar y teñir las estructuras duras antes de realizar un estudio de edad y crecimiento y así incrementar la confiabilidad de las estimaciones de edad.

 KEYWORDS age growth sharks batoids freshwater stingray precision bias PALABRAS CLAVE edad crecimiento tiburones rayas raya de agua dulce precisión sesgo INTRODUCTION

Due to their life history characteristics (Cortés et al. 2012) and the increase in fishing pressure, elasmobranchs are currently one of the most vulnerable fish groups, with high threat levels worldwide (Stevens et al. 2000, Dulvy et al. 2014). Age estimation for these species is therefore important to suggest fishery management measures. This information is also relevant for the estimation of growth and mortality rates, maturity age and longevity, among other parameters, as they are primary inputs in demographic studies that allow the vulnerability and productivity of the populations to be established (e.g. Campana 2001, Goldman et al. 2012). Despite their importance, the number of age and growth publications in tropical elasmobranchs is relatively low (14.1%; 25 of 177 reviewed papers; e.g. Harry et al. 2010, O’Shea et al. 2013, Mejía-Falla et al. 2014) compared with those in cold and temperate waters (133 papers; e.g. Duman and Başusta 2013, James et al. 2014, Kadri et al. 2013) (Supplementary material Table S1).

Age studies in elasmobranchs are based on counting pairs of growth bands in hard structures (e.g. vertebrae, spines) which are formed periodically. As the visualization of these band pairs in some cases is not simple, over time several methods, such as X-rays (Natanson and Cailliet 1990), staining (Goldman et al. 2012) and histological sections (Natanson et al. 2007), have been suggested and tested to enhance the visualization of the growth bands and therefore improve the accuracy of age estimation (Goldman et al. 2012).

Of the aforementioned processes, staining has been the most widely used, mainly because it is the least complex and least expensive procedure, and it is even suggested as a first step before applying more demanding and expensive methods (Goldman et al. 2012). Staining techniques have been used in age studies of several elasmobranch species (e.g. Neer and Cailliet 2001, Fernández-Carvalho et al. 2011, Torres-Palacios et al. 2019). The result of each technique has been found to be species-specific (Goldman et al. 2012), so it is not possible to define a standard protocol for the use of these techniques in elasmobranchs.

Furthermore, few studies have reported in detail the procedures applied to select the most appropriate staining method for each species (e.g. Fernández-Carvalho et al. 2011, Huveneers et al. 2013, Torres-Palacios et al. 2019), and a large number of studies have applied methods previously described in similar species without evaluating their effectiveness in the particular study species (e.g. Aversa et al. 2011, Sánchez de Ita et al. 2011, Chin et al. 2013). Given that there is evidence that the results of these techniques are species-specific, the need to assess and test how these tools influence the visualization of the growth bands before carrying out age and growth studies is highlighted. Additionally, recent studies have shown that band formation in some elasmobranch species occurs bi-annually (Wells et al. 2013) or irregularly (Huveneers et al. 2013), so their age may be underestimated (Hamady et al. 2014, Harry 2018, Natanson et al. 2018). Therefore, improving the visualization of the bands and facilitating their counting would reduce the sources of error and increase the precision of the readings.

For the above reasons, and because studies based on radiocarbon dating, histology and X-rays are more restrictive in tropical countries, this study aimed to evaluate different staining techniques in vertebrae of nine tropical elasmobranch species to determine those that facilitate the visualization and counting of the growth bands, contributing to future age estimates and population assessments of the evaluated tropical species.

 MATERIALS AND METHODS Vertebrae processing

A total of 428 individuals were collected between 2007 ans 2015 from fisheries of the Colombian Pacific region (79°44′W, 5°45′N; 77°12′W, 2°15′N). These individuals belong to four batoid species, Narcine leoparda (n=90 individuals), Urotrygon aspidura (n=90 individuals), Hypanus longus (n=90 individuals) and Potamotrygon magdalenae (n=24 individuals); and five shark species, Alopias pelagicus (n=27 individuals), Carcharhinus falciformis (n=21 individuals), Sphyrna lewini (n=53 individuals), Sphyrna corona (n=18 individuals) and Mustelus lunulatus (n=15 individuals).

Once each specimen was identified at the species level, its sex was determined and total length of sharks or disc width of rays was recorded. From each individual, a section of 14 to 16 vertebrae was extracted from under the first dorsal fin (in sharks) and from the abdominal region (in rays), which were labelled and frozen until laboratory analysis. The excess tissue in the vertebrae was removed using a scalpel. Because affinity of the stains and visualization of the growth bands could be affected by the individual size, vertebrae were separated into three size intervals: small, medium and large diameter. This was done for the species whose sample size allowed it.

As the thickness of the vertebrae sections influences the visualization and reading of the growth bands, size ranges of the vertebrae with different section thicknesses (0.3 to 0.7 mm) were combined per individual. For this purpose, each vertebra was fixed to a slide with Crystalbond 509 and cut sagittally with an Isomet Buehler low-speed saw cutter with two diamond head blades (Buehler, Lake Bluff, IL, USA) to obtain bow-tie sections (Cailliet and Goldman 2004). The thickness of the section that best allowed the visualization of the bands was based on a qualitative evaluation carried out by expert readers.

In order to assess the effect of the stains used, sections were randomized and stained with alizarin red (0.05%), methylene blue (0.001%), crystal violet (0.001%), basic fuchsin (0.001%), acid fuchsin (0.001%), Bismarck brown (0.05%), light green (0.05%), silver nitrate (1%) and the Dahl staining (alizarin red 0.01% and light green 0.05%). Each dye was applied in successive intervals of one minute until reaching its saturation point in each structure. This was done as a preliminary test for a sub-sample of each species (n=5).

Subsequently, the best three staining times for each dye were qualitatively established and applied to the vertebrae of the individuals selected for the analysis. Biases given by individual variations were avoided using several vertebrae per individual to apply the treatments (stain + staining time). Sections (with and without staining) were mounted on a slide, observed under a microscope using transmitted light and photographed for each treatment. Additionally, vertebral sections without any staining were used to evaluate the effect of immersion oil and distilled water (imbibing each in a drop of the substance) in the visualization of the growth bands. The images obtained were analysed with the Image Pro Plus 7.0 software (Media Cybernetics) in which two skilled readers performed the quantitative counting of the growth bands independently in each sample. Readings were carried out twice per reader, who did not know the details of the sex, size or previous reading of each vertebra. Based on this information, analyses were carried out to establish the accuracy and bias among readers and subsequently to establish the most efficient technique for observing and counting growth bands per species.

 Data analysis

In order to assess the degree of precision in the vertebral band readings among readers, the index of average percentage error, the coefficient of variation, the percentage of agreement between readers, Bowker’s symmetry test and the percentage of vertebrae read were calculated and analysed as follows.

The index of average percentage error (IAPE) provided information on the accuracy of age estimations among readers; small values indicated more precise readings (Beamish and Fournier 1981). The IAPE was calculated as follows:

IAPE=[ 1 n ( 1 R ∑ i=1 R | x ij − x ¯ j | x ¯ j ) ]×100

where n is the number of samples, xij is the ith age estimation for individual j, R is the number of readings and Xj is the average age calculated for individual j.

Coefficient of variation (CV) measured reading accuracy (Campana 2001) expressed as the proportion of the mean and standard deviation, as follows:

CV=[ 1 n ( ∑ i=1 R ( x ij − x ¯ j ) 2 R x ¯ j ) ]×100

where xij is the ith age for the individual j, is the average age of individual j and R is the number of readers.

Percentage of agreement between readers (PA) allowed us to establish the variation in the reading of bands among readers (Goldman 2002), using the following equation that was applied to each vertebra size range (diameter) and considering differences between bands (0, ±1, ±2).

PA=( number of agreements in the reading total number of reading )100

Bowker’s symmetry test determined, using a chi-square test, whether differences between the readers were systematic (p<0.05) or random (p>0.05; Hoenig et al. 1995), the latter being the expected result because random errors indicate that there is no bias among readers.

Percentage of vertebrae read (RV) estimated the proportion of vertebrae that could be read successfully considering the entire sample; higher values indicated better performance of the assessed technique.

An evaluation of the results of all the tests applied was carried out in order to determine the most efficient technique for observing and estimating the age in each species studied. Subsequently, the final decision per species was taken by comparing the results of each of the treatments as a whole and by vertebrae size.

 RESULTS Section thickness

The qualitative evaluation of the section thickness for batoids showed that sections of 0.4 mm were most suitable, except for Narcine leoparda, for which, as for all the shark species assessed, sections of 0.5 mm are recommended. These thicknesses allowed the growth bands to be visualized and counted more easily in the three size classes assessed (large, medium and small) for Narcine leoparda, Urotrygon aspidura and Hypanus longus. In addition to the visibility of the bands, this thickness showed the lowest proportion of fractured vertebrae during the sectioning process. The other thicknesses evaluated did not allow the clear observation of the growth bands because of the low contrast or excess light passage (0.3 mm) or, on the contrary, because of too little light passage through the sections (0.6 and 0.7 mm).

 Batoids

Narcine leoparda (n=90 individuals)

For the leopard electric ray, treatments were applied for three vertebrae size intervals: small (diameters of 1.30 to 2.13 mm), medium (2.14 to 2.99 mm) and large (3.00 to 4.84 mm). There was no systematic bias in any of the treatments used for visualization of growth bands, except for small vertebrae stained with alizarin red for 14 min (Supplementary material Table S2).

Treatment analysis by vertebrae size showed that the highest PA (±0 bands) values were found in unstained large vertebrae (91.3%), medium vertebrae stained with basic fuchsin for 7 min (73.1%), and small vertebrae stained with methylene blue for 1 min (75%) followed by alizarin red for 16 min (73.1%) (Table S2). The three vertebrae sizes evaluated showed percentages of read vertebrae (RV) higher than 70% in all treatments. The highest RV values were found for large vertebrae stained with methylene blue and basic fuchsin for 3 min (90%); for medium vertebrae without staining or immersed in oil, basic fuchsin for 7 min or alizarin red for 14 min (86.7%); and for small vertebrae immersed in distilled water or in alizarin red for 14 and 16 min (86.7%) (Table S2). IAPE and CV showed relatively low values in large and medium vertebrae and high values in small vertebrae, except in those stained with alizarin red for 16 min, which showed the best values for small and medium intervals.

In conclusion, unstained large vertebrae and the medium and small vertebrae treated with alizarin red for 16 min (Fig. 1A-C) showed the best combination of values in the precision and bias tests between readers. Further, these treatments obtained the lowest IAPE and CV, the highest total agreement percentage and a high percentage of read vertebrae (Table 1, S2).

Vertebra sections showing the best treatment (stain and staining time) for each batoid species. The scale bar corresponds to 1 mm. Results of the precision and bias tests between readers for selected treatments for small, medium and large vertebrae of <italic>Narcine leoparda, Urotrygon aspidura </italic>and<italic> Hypanus longus</italic>, and for all vertebrae of <italic>Potamotrygon magdalenae, Alopias pelagicus, Carcharhinus falciformis, Sphyrna lewini, Sphyrna corona </italic>and <italic>Mustelus lunulatus</italic>. IAPE, index of average error percentage; CV, coefficient of variation; PA (±0 bands), percentage of total agreement; RV, percentage of read vertebrae; p, p-values of Bowker’s symmetry test. Dash indicates that data were not available because the treatment was not used.
Species Vertebra size Treatment (stain + staining time) IAPE CV PA RV p
Narcine leoparda Small Alizarin red 16′ 4.9 7.0 73.1 86.7 0.14
Medium Alizarin red 16′ 3.3 4.6 64.0 83.3 0.22
Large No staining 0.7 1.7 91.3 76.7 0.09
Urotrygon aspidura Small Light green 5′ 5.1 7.3 64.7 76.5 0.16
Medium Methylene blue 20′ 1.2 1.7 84.2 89.5 0.32
Large Methylene blue 10′ 2.2 3.1 80.0 100.0 0.39
Hypanus longus Small Bismarck brown 1′ 2.2 3.1 93.3 100 0.32
Bismarck brown 2.0′ 2.2 3.1 93.3 100 0.32
Light green 0.5′ 2.2 3.1 93.3 100 0.32
Light green 1′ 2.2 3.1 93.3 100 0.32
Medium Basic fuchsin 2′ 2.2 3.1 93.3 100% 0.32
Large Light green 7′ 0.0 0.0 100.0 100.0 –
Potamotrygon magdalenae All Alizarin red 7′ 0.1 1.3 95.5 91.7 0.16
Alopias pelagicus All Bismarck brown 15′ 1.8 2.5 57.1 77.8 0.39
Carcharhinus falciformis All Crystal violet 35′ 0.5 0.7 85.7 63.6 0.42
Sphyrna lewini All Immersion oil 7.0 2.4 89.8 94.2 0.14
Sphyrna corona All Crystal violet 20′ 0.0 0.0 100.0 55.6 0.16
Mustelus lunulatus All Light green 40′ 0.0 0.0 100.0 78.6 0.30

Urotrygon aspidura (n=90 individuals)

For the Panamic stingray, treatments were also applied to three vertebrae size: small (diameters of 0.70 to 1.59 mm), medium (1.60 to 2.49 mm) and large (2.50 to 3.30 mm). Only the treatment with basic fuchsin for 1 min in medium vertebrae showed bias in the reading; for all others, the differences were due to random errors (Table S3).

The percentage of read vertebrae was higher than 70% in all treatments for the three size interval assessed, reaching a maximum of 100% with methylene blue for 10 min and alcohol in small vertebrae, with alizarin red for 15 min in medium vertebrae, and with almost all the treatments in the large vertebrae, except with crystal violet (Table S3). The highest PA (±0 bands) between readers occurred with alizarin red for 15 min in small vertebrae, methylene blue for 20 min in medium vertebrae, and methylene blue for 10 min in large vertebrae.

The lowest CV and IAPE values were found with methylene blue for 10 min in large vertebrae (Fig. 1D), with methylene blue for 20 min in medium vertebrae (Fig. 1E), and with light green for 5 min in small vertebrae (Fig. 1F). These stains also showed a high percentage of total agreement and a high percentage of read vertebrae, being chosen as the best treatment for each vertebra size (Tables 1, S3).

Hypanus longus (n=90 individuals)

Vertebra sections of H. longus were also separated in three size intervals: small (diameters of 3.00 to 5.15 mm), medium (5.16 to 8.39 mm) and large (8.40 to 12.34 mm). For this species, four stains were discarded (methylene blue, crystal violet, immersion oil and acid fuchsin) as the vertebral growth bands showed no clear delimitation among them, and this would increase the reading errors.

PA values varied between 80.0% (no staining, basic fuchsin for 1 min and light green for 2 min) and 100% (light green for 7 min) in large vertebrae; between 53.9% (basic fuchsin for 3 min) and 93.3% (basic fuchsin for 2 min) in medium vertebrae, and from 73.3% (basic fuchsin for 1 and 3 min) to 93.3% (Bismarck brown for 1 and 2 min, light green for 0.5 and 1 min) in small vertebrae (Table S4). Previously selected stains showed variable IAPE and CV values within and among vertebrae sizes; the lowest values were found for small vertebrae stained with Bismarck brown for 1 and 2 min and light green for 0.5 and 1 min, for medium vertebrae stained with Bismarck brown for 2 and 3 min, and for large vertebrae stained with light green for 7 min (Table S4). In general, vertebrae with no treatment showed higher IAPE and CV values than vertebrae that received staining. All vertebrae sections of this species were read (100% RV in all cases).

Considering all the results, the best treatment was light green for 7 min for large vertebrae of H. longus, basic fuchsin for 2 min for medium vertebrae (although Bismarck brown for 2 and 3 min were also good treatments), and Bismarck brown for 1 and 2 min and light green for 0.5 and 1 min for small vertebrae (Table S4).

Potamotrygon magdalenae (n=24 individuals)

Age bias analysis of Magdalena river stingray showed no systematic bias in readings or treatments (Table S5). The percentage of read vertebrae varied between 52.9% for light green for 40 min and 95% for methylene blue for 30 and 40 min, the latter being followed by alizarin red for 7 min, with 91.7%. The lowest IAPE and CV values and the highest PA (±0 bands) values between readers were found with crystal violet for 60 min and light green for 40 min (0 IAPE, 0 CV, 100% PA each), followed by alizarin red for 7 min (0.1 IAPE, 1.3 CV, 95.5% PA). The group analysis of the treatments showed that vertebrae treated with light green and crystal violet require too much time for staining (≥40 min), while alizarin red showed the second-best values, with only 7 min of staining (Tables 1, S5; Fig. 1J).

 Sharks

Alopias pelagicus (n=27 individuals)

Vertebrae of the pelagic thresher shark involved high difficulty in observing and counting a growth band pattern, generating high variation in the precision tests and percentages of read vertebrae (Table S5). None of the treatments showed systematic bias in their readings (P>0.05 in all cases). Vertebrae stained with alizarin red for 5 and 7 min showed the best results in the precision analyses (IAPE, CV and PA), but the percentages of RV with these treatments were very low. Conversely, basic fuchsin for 45 min showed the highest RV (91.7%) but a low PA value (±0 bands=36.4%). Considering the values of all the tests, staining with Bismarck brown for 15 min showed a good performance, occupying the third place in IAPE, CV, and RV and the fourth place in PA values (Tables 1, S5; Fig. 2A).

Vertebra sections showing the best treatment (stain and staining time) for each shark species. The scale bar corresponds to 1 mm.

Carcharhinus falciformis (n=21 individuals)

None of the treatments analysed for the silky shark showed systematic bias in the readings (P>0.05 for all cases). The highest PA (±0 bands) values between readers were found with silver nitrate for 2 and 3 min (100%) and crystal violet for 35 min (85.7%). Similarly, the lowest IAPE and CV values were obtained with silver nitrate for 2 and 3 min, crystal violet for 35 min and acid fuchsin for 50 min. However, the percentage of RV ranged from a very low value (16.7%) for silver nitrate for 2-3 min to 100% for light green for 35 min (Table S5). Vertebrae stained with crystal violet for 35 min showed the second-best values of IAPE (0.5), CV (0.7) and PA (85.7%), as well as an acceptable RV value (63.6%), being considered the most appropriate treatment for this species (Table 1, Fig. 2B). The vertebrae without staining showed the lowest performance in the precision tests, followed by methylene blue for 25 min and basic fuchsin for 40 min (Table S5).

Sphyrna lewini (n=52 individuals)

For the scalloped hammerhead none of the treatments analysed showed systematic bias in the readings (except using Bismarck’s brown for 25 min, P=0.02; Table S6), and all showed a high percentage of RV (>84%) but also relatively high IAPE and CV values. The best values of PA (±0 bands), IAPE and CV were found with immersion oil and methylene blue for 35 and 30 min, which was the best treatments for the species. Therefore, immersion oil was suggested as the reagent for treating the vertebrae of S. lewini (Table 1, Fig. 2C).

Sphyrna corona (n=18 individuals)

For the scalloped bonnethead none of the treatments analysed showed systematic bias in the readings (P>0.05 in all cases) and all had a percentage of RV higher than 50%, reaching a maximum of 77.8% (with immersion oil and light green for 35 min). The highest PA values (±0 bands) were found with crystal violet for 20 min (100%), methylene blue for 25 min (81.8%) and crystal violet for 25 min (80.0%; Table S6); similarly, IAPE and CV were lower with crystal violet for 20 and 25 min, as well as with methylene blue for 25 min. Vertebrae treated with crystal violet for 20 min showed the best combination of precision and bias values, establishing it as the best treatment for this species (Table 1, Fig. 2D).

Mustelus lunulatus (n=15 individuals)

For the smooth-hound shark, none of the treatments analysed showed systematic bias in the readings (P>0.05) and the percentage of RV was higher than 60% in all treatments, reaching a maximum of 100% with crystal violet for 30 and 40 min. The highest PA values (±0 bands) and lowest IAPE and CV values were found with light green for 40 min (100%; 0 and 0, respectively) and 30 min (91.7%; 1.2 and 1.7, respectively), followed by crystal violet for 20 min (71.4%; 3.2 and 4.5; Table S6). From the group analysis of the treatments assessed, light green for 40 min showed the best combination of values and is therefore the most appropriate treatment for the species (Table 1, Fig. 2E).

 DISCUSSION

There is sufficient evidence that the visualization of the growth bands in hard structures of elasmobranchs varies substantially among species (Duarte et al. 2001, Goldman et al. 2012, this study). Therefore, it is essential to identify and apply species-specific techniques that enhance this visualization and consequently increase the accuracy of age and growth estimations. In this study, the results support this statement for the marine species analysed, which come from a single geographical region, revealing that alizarin red works better for N. leoparda, light green for M. lunulatus, immersion oil for S. lewini, Bismarck brown for A. pelagicus, crystal violet for S. corona and C. falciformis, methylene blue and light green for U. aspidura and light green, basic fuchsin and Bismarck brown for H. longus, Furthermore, our research documents intraspecific variation according to the size of the vertebrae in U. aspidura and H. longus (see Supplementary material, Species comments section). Similarly, vertebrae staining with alizarin red works better for the freshwater stingray P. magdalenae.

Additionally, systematic development of sectioning and staining of the vertebrae and visualizing and counting the growth bands with different arrangements to establish the best combination is a great training exercise for the researchers, which will result in a higher precision in identifying and performing growth band counts and therefore a better estimation of the age of an individual.

Despite the evidence on the importance of developing this procedure by species to establish age and growth of elasmobranchs, many papers do not include detailed information on the procedures performed. This may be due to limited word constraints in publications (usually limiting them to one sentence) or to the fact that these procedures were not performed (and a thickness-stain-staining time combination already established in other studies was chosen; e.g. Kadri et al. 2013, O’Shea et al. 2013, Drew et al. 2015). A review of publications on elasmobranch age and growth (n=177) showed that only 39% used staining techniques; of these, 40 were carried out on rays and 29 on sharks (Table S1). Furthermore, the number of studies regarding precision and bias evaluations to compare treatments is even smaller.

Although the bias in the band reading was almost null, the greatest difficulty in the counts was related to the definition of the birthmark and the reading of the bands located towards the edges of the vertebrae. Consequently, the greatest differences between readers occurred in areas near the focus and on the edge of the vertebral sections. Other studies that used similar methods in sagittal vertebrae sections have experienced problems with sharpness in these same areas (e.g. Licandeo et al. 2006, McFarlane and King 2006). Similarly, Ainsley et al. (2011) recorded difficulties for identifying band pairs close to the focus in about 20% of the individuals of Amblyraja radiata, Malacoraja senta and Bathyraja interrupta. Band reading problems in the distal region of the vertebrae, especially in older specimens, could be due to the proximity of the last and penultimate band, or to the fact that growth zone periodicity changes or ceases later in life, potentially after the onset of sexual maturity (Harry 2018, Natanson et al. 2018). The cited authors proposed several effects generated by those underestimates and the techniques available to address them.

There are also contrasting results with those found in this study regarding cut thickness, since wider or thinner thicknesses have been proposed as the most suitable for counting growth bands in other species such as Mustelus canis, Mustelus asterias, Prionace glauca and Pristis pectinata (e.g. Conrath et al. 2002, Farrell et al. 2010). This implies that the suitable visualization of bands with a certain section thickness is not a shared attribute in all elasmobranch species, even in species of the same genus, as was the case in Bathytoshia centroura (formerly Dasyatis centroura) and Dasyatis pastinaca (Yigin and Ismen 2012). In these species, the section thicknesses chosen by the authors were 0.6 mm and 0.5 mm, respectively, which differ from the optimum found for Hypanus longus (formerly Dasyatis longa) in this study (0.4 mm).

These procedures that require few logistical resources and significant investment of time, substantially improve the results obtained in terms of experience, learning and quality of the data. However, as seen in this study, this is not a general rule, and some shark and ray species have vertebral structures that can be easily visualized without staining procedures (e.g. large vertebrae of N. leoparda), while for others staining are good treatment (e.g. C. falciformis). As another example, Geraghty et al. (2013) found no differences in the accuracy of the age estimate of Carcharhinus brevipinna, C. obscurus and C. plumbeus among unstained vertebrae and those stained with alizarin red and crystal violet. Whatever the case may be, it is suggested that the usefulness of the staining tests should be tested and evaluated through specific quantitative analyses, as shown in this study.

From the results obtained in this study we conclude that it is essential to consider several precision indexes (PA, IAPE, and CV) to define, with the best possible criteria, the combination of section thickness, stain and staining time that is most suitable for the species of interest (Supplementary material, Species comments section). It is a mistake to make this decision based on a single index and to assess the performance of a single technique without contrasting or comparing treatments. In this regard, most studies that used staining techniques have found better performance with stained vertebrae than without staining. Duarte et al. (2001) found better precision and bias values in age estimation of Galeorhinus galeus using cobalt nitrate; Girgin and Başusta (2016) found better results for Dasyatis pastinaca using safranin-O than using crystal violet, silver nitrate and alcian blue. Furthermore, these authors found differences in the sizes within the age groups estimated for the species from those found by Ismen (2003), assigning these differences to the staining technique used. Additionally, Başusta et al. (2017) suggested the use of crystal violet to improve visualization in Raja clavata, safranin-O in Raja asterias, Gymnura altavela and Torpedo marmorata and alcian blue in Raja miraletus and Rhinobatos rhinobatos, also finding differences among genera.

All the examples mentioned above illustrate how the implementation of techniques to enhance the visualization of growth bands significantly influences the accuracy of the age estimation, thus supporting our results. Even the results obtained versus the bibliographic references reviewed show how studies carried out within a same species in different geographical locations (latitudinal differences) identified different techniques as the most appropriate. Furthermore, considering only tropical species, a great variation is identified in the techniques selected for the visualization of the growth bands. This reaffirms the importance of evaluating different combinations (thickness, stain, staining time) for each species to be studied (Table 2). The conditions that produce these intraspecific variations are unknown, but may involve environmental factors (temperature and/or productivity) or be related to physiological changes induced by the consumption of food and starvation periods, which cause variation in salt deposition and consequently in the formation of pairs of growth bands (Goldman 2005).

Age and growth studies of tropical distribution elasmobranch species, indicating the techniques used to visualize the vertebral growth bands and the technique selected for each species. *Studies that include tropical and subtropical areas.
Species Study area Techniques used Stainings used Technique defined Reference
Batoids
Dasyatis lata Kane’ohe Bay on Oahu, Hawai’i, USA Sagittal sectioning, OTC None Sagittal sectioning Dale and Holland (2012)
Glaucostegus typus Cleveland Bay, Great Barrier Reef, Australia Sagittal sectioning None 0.4 - 0.6 mm sagittal sectioning White et al. (2014)
Himantura uarnak Ningaloo Reef Marine Park, Australia Sagittal sectioning None 0.35 mm sagittal sectioning O’Shea et al. (2013)
Hypanus guttatus Eastern Atlantic coast, Rio Grande do Norte, Brazil Sagittal sectioning None 0.2 mm sagittal sectioning Gianeti et al. (2019)
Neotrygon annotata North-east Australia Sagittal sectioning None 0.2 - 0.3 mm sagittal sectioning. Jacobsen and Bennett (2010)
Neotrygon kuhlii Ningaloo Reef Marine Park, Australia Sagittal sectioning None 0.35 mm sagittal sectioning O’Shea et al. (2013)
North-east Australia Sagittal sectioning None 0.2 - 0.3 mm sagittal sectioning. Jacobsen and Bennett (2010)
Neotrygon picta North-east Australia Sagittal sectioning None 0.2 - 0.3 mm sagittal sectioning Jacobsen and Bennett (2010)
Pastinachus atrus Ningaloo Reef Marine Park, Australia Sagittal sectioning None 0.35 mm sagittal sectioning O’Shea et al. (2013)
Rhynchobatus australiae Cleveland Bay, Australia Sagittal sectioning None 0.4 - 0.6 mm sagittal sectioning White et al. (2014)
Rhynchobatus laevis Cleveland Bay, Australia Sagittal sectioning None 0.4 - 0.6 mm sagittal sectioning White et al. (2014)
Rhynchobatus palpebratus Cleveland Bay, Australia Sagittal sectioning None 0.4 - 0.6 mm sagittal sectioning White et al. (2014)
Taeniura lymma Ningaloo Reef Marine Park, Australia Sagittal sectioning None 0.35 mm sagittal sectioning O’Shea et al. (2013)
Urotrygon aspidura Central Pacific coast, Colombia Sagittal sectioning and staining Light green (0.05%)
methylene blue (0.001%) 		0.4 mm sagittal sectioning staining using light green for small vertebrae and methylene blue for medium and large vertebrae Torres-Palacios et al. (2019)
Urotrygon chilensis Tehuantepec Gulf, Southeast Pacific, Mexico Sagittal sectioning None 0.3 - 0.5 mm sagittal sectioning Guzmán-Castellanos (2015)
Urotrygon microphthalmum Eastern Atlantic, Pernambuco, Brazil Sagittal sectioning None 0.3 mm sagittal sectioning Santander-Neto (2015)
Urotrygon rogersi Central Pacific coast, Colombia Sagittal sectioning Several
(not indicated) 		0.4 mm sagittal sectioning Mejía-Falla et al. (2014)
Sharks
Alopias pelagicus Java Sea, Indonesia Sagittal sectioning None 0.3 mm sagittal sectioning Drew et al. (2015)
Alopias superciliosus Northeastern Taiwan X-ray radiography and staining Silver nitrate X-ray radiography Liu et al. (1998)
Atlantic Ocean* Sagittal sectioning and staining Crystal violet 0.5 mm sagittal sectioning and staining Fernández-Carvalho et al. (2015)
Carcharhinus coatesi Queensland, Australia* Sagittal sectioning None 0.4 mm sagittal sectioning Smart et al. (2012)
Carcharhinus fitzroyensis Queensland, Australia* Sagittal sectioning None 0.4 mm sagittal sectioning Smart et al. (2012)
Carcharhinus leucas Veracruz and Campeche, Mexico Sagittal sectioning None 0.3 - 0.6 mm sagittal sectioning Cruz-Martínez et al. (2005)
Carcharhinus limbatus Eastern Lombok, Indonesia Sagittal sectioning None 0.4 mm sagittal sectioning Smart et al. (2015)
Carcharhinus macloti Queensland, Australia* Sagittal sectioning None 0.4 mm sagittal sectioning Smart et al. (2012)
Carcharhinus plumbeus Northeastern Taiwan Staining of sagittal and longitudinal sectioning, X-radiography of sagittal and longitudinal sectioning Eosin, haematoxylin X-radiography Joung et al. (2004)
Carcharhinus porosus Eastern Atlantic coast, Maranhão, Brazil Sagittal sectioning, staining and cedarwood oil Alizarin red
cedarwood oil 		Sagittal sectioning and staining Lessa and Santana (1998)
Carcharhinus sorrah Northern Australia Staining. Protein stains mercurochrome and ninhydrin.
Histology, Radiography
X-ray, spectrometry, image analysis and examination of sectioned vertebrae under transmitted, reflected, interference and polarized light OTC 		Silver nitrate, Alizarin Red S, crystal violet
cobalt nitrate, ammonium sulphide, mercurochrome ninhydrin 		Staining with ninhydrin Davenport and Stevens (1988)
Carcharhinus tilstoni Northern Australia Staining. Protein stains mercurochrome and ninhydrin.
Histology, Radiography
X-ray, spectrometry, image analysis and examination of sectioned vertebrae under transmitted, reflected, interference and polarized light OTC 		Silver nitrate, Alizarin Red S, crystal violet, cobalt nitrate, ammonium sulphide, mercurochrome and ninhydrin Staining with ninhydrin Davenport and Stevens (1988)
Eusphyra blochii Queensland, Australia* Sagittal sectioning None 0.4 mm sagittal sectioning Smart et al. (2012)
Galeocerdo cuvier Australian east coast* Sagittal sectioning and staining Unstained, crystal violet, silver nitrate 0.15 mm sagittal sectioning Holmes et al. (2015)
Hemipristis elongata Queensland, Australia* Sagittal sectioning None 0.4 mm sagittal sectioning Smart et al. (2012)
Isogomphodon oxyrhynchus Eastern Atlantic coast, Maranhão, Brazil Sagittal sectioning and staining Alizarin Red S Sagittal sectioning and staining Lessa et al. (2000)
Isurus oxyrinchus Western and central North Pacific Ocean* Shadowing (half-cut centra)
staining, soft X-radiography (whole or half-cut centra) 		Alizarin red, silver nitrate Shadowing on half-cut centra Semba et al. (2009)
Western and central Atlantic* Sagittal sectioning None 0.3 mm sagittal sectioning Barreto et al. (2016)
Southern Indian Ocean* Sagittal sectioning
and soft X-ray 		None 1-1.44 mm sagittal sectioning and soft X-ray Liu et al. (2018)
Prionace glauca Eastern Atlantic, Brazil Sagittal sectioning None 1 mm sagittal sectioning Lessa et al. (2004)
Baja California Peninsula, Mexico Sagittal sectioning
and staining 		Silver nitrate Whole vertebra stained with silver nitrate and 0.5 mm sagittal sectioning Blanco-Parra et al. (2008)
South Pacific Ocean* Transversal sectioning
and soft X-ray radiograph 		None Transversal sectioning
and soft X-ray radiograph 		Joung et al. (2018)
Rhincodon typus* Northwestern Pacific, Taiwan* Transversal sectioning and X-ray radiograph None Transversal sectioning and X-ray radiograph Hsu et al. (2014)
Northwestern Pacific, Taiwan and Pakistan* Sagittal sectioning
Bomb radiocarbon 		None Sagittal sectioning
Bomb radiocarbon 		Ong et al. (2020)
Rhizoprionodon acutus Northeastern coast, Australia Sagittal sectioning None 0.4-0.6 mm sagittal sectioning Harry et al. (2010)
Western Atlantic, Senegal Sagittal sectioning
and staining 		Acetic acid + Toluidine blue Sagittal sectioning and staining Ba et al. (2015)
Rhizoprionodon lalandii Eastern Atlantic coast, Maranhão, Brazil Sagittal sectioning None 0.3 mm sagittal sectioning Lessa et al. (2009)
Rhizoprionodon porosus Eastern Atlantic coast, Maranhão, Brazil Sagittal sectioning None 0.3 mm sagittal sectioning Lessa et al. (2009)
Rhizoprionodon taylor Cleveland Bay, Australia Vertebrae grounding None 0.2-0.4 mm grounding Simpfendorfer (1993)
Sphyrna lewini Northeastern Taiwan Sagittal sectioning None 0.2 mm sagittal sectioning Chen et al. (1990)
Michoacan, Mexico Sagittal sectioning
and staining 		Crystal violet (0.01%) Sagittal sectioning and staining Anislado-Tolentino and Robinson-Mendoza (2001)
Java Sea, Indonesia Sagittal sectioning None 0.3 mm sagittal sectioning Drew et al. (2015)

This variation could determine the calcification of the vertebrae and the affinity of the stains to these structures (based on the carbonate and/or phosphate concentrations), influencing the nature of the dye (basophilic or acidophilic) on the quality of the dye and hence the visualization of the growth bands. However, it is recommended to further this analysis in order to establish whether there is any influence of the type of dye on its ability to improve the visibility of the growth bands. In this regard, it is necessary to carry out a study on the possible causes of the accumulation and type of compounds that make possible changes in birthmarks, e.g. ontogenetic changes in the diet, temperature or reabsorption of materials accumulated in the vertebrae (Licandeo et al. 2006).

Parameter estimation from age and growth studies has profound implications in population assessments based on demography, directly affecting the estimation of demographic parameters and thus the potential management of the species based on their life history traits. Hence, an incorrect specification of the bands and their deposition frequency could lead to the under- or overestimation of the growth coefficient and the asymptotic lengths of the populations. Therefore, any effort made to reduce the bias in the visualization, counting and analysis of the growth bands is an indirect but essential contribution to the management and subsequent conservation of elasmobranch species.

 ACKNOWLEDGEMENTS

This study was financed by the Departamento Administrativo de Ciencia, Tecnología e Innovación - Colciencias (Contract No: RC 156-2010), PADI Foundation, and Iniciativa de Especies Amenazadas (Scholarship IEA-2012-04-12-18-55-26).

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The following supplementary material is available through the online version of this article and at the following link:
http://scimar.icm.csic.es/scimar/supplm/sm05045esm.pdf

Table S1. – Age and growth publications in tropical elasmobranchs (Spreadsheet in MS Excel format available at: http://scimar.icm.csic.es/scimar/supplm/sm05045TableS1.xlsx).

Table S2. – Results of precision and bias tests between readers, for each of the treatments applied to the small, medium and large vertebrae of Narcine leoparda. IAPE, index of average error percentage; CV, coefficient of variation; PA (±0 bands), percentage of total agreement; RV, percentage of read vertebrae; P, p-values of the Bowker symmetry test. Best results of each test for each vertebrae size are highlighted in bold and the selected treatments are shaded in gray.

Table S3. – Results of precision and bias tests between readers, for each of the treatments applied to the small, medium and large vertebrae of Urotrygon aspidura. IAPE, index of average error percentage; CV, coefficient of variation; PA (±0 bands), percentage of total agreement; RV, percentage of read vertebrae; P, p-values of the Bowker symmetry test. Best results of each test for each vertebrae size are highlighted in bold and the selected treatments are shaded in gray.

Table S4. – Results of precision and bias tests between readers, for each of the treatments applied to the small, medium and large vertebrae of Hypanus longus. IAPE, index of average error percentage; CV, coefficient of variation; PA (±0 bands), percentage of total agreement; RV, percentage of read vertebrae; P, p-values of the Bowker symmetry test. Best results of each test for each vertebrae size are highlighted in bold and the selected treatments are shaded in gray.

Table S5. – Results of precision and bias tests between readers for each of the treatments applied to Potamotrygon magdalenae, Alopias pelagicus and Carcharhinus falciformis vertebrae. IAPE, index of average error percentage; CV, coefficient of variation; PA (±0 bands), percentage of total agreement; RV, percentage of read vertebrae; P, p-values of the Bowker symmetry test. Best results of each test are highlighted in bold and the selected treatment for each species.is shaded in gray.

Table S6. – Results of precision and bias tests between readers for each of the treatments applied to Sphyrna lewini, Sphyrna corona and Mustelus lunulatus vertebrae. IAPE, index of average error percentage; CV, coefficient of variation; PA (±0 bands), percentage of total agreement; RV, percentage of read vertebrae; P, p-values of the Bowker symmetry test. Best results of each test are highlighted in bold and the selected treatment for each species.is shaded in gray.

Species comments.