Reconstructing the Historical Density, Size, and Age Structure of the Noble Pen Shell (Pinna nobilis) Population: Insights from Malo Jezero Lagoon, Mljet National Park (Adriatic Sea)

Abstract

The noble pen shell (Pinna nobilis) is a key bivalve species found in the Mediterranean that has suffered dramatic declines due to mass mortality events (MMEs) caused by pathogens like Haplosporidium pinnae. This study looks at the historical population structure of P. nobilis in Malo jezero, a coastal lagoon in Mljet National Park, Croatia, using data collected before the MME. During a field survey in 2018, data on the population density, size, and age of 3800 individuals, using a grid-based transect method, were collected. The population density ranged from 7.50 to 55.83 individuals per 100 m², with an average of 25.42 individuals per 100 m², over 11 520 m², reflecting a high abundance compared to other populations. All individuals were mature, with no juveniles or signs of recent recruitment. The estimated ages ranged from 8.00 to 44.34 years, with 20 individuals exceeding the expected maximum size. The population was comprised of older individuals, making it vulnerable to sudden events, due to a lack of younger individuals. The isolation of Malo jezero may limit larval exchange with other populations, contributing to recruitment challenges. This study provides important information for understanding P. nobilis populations and supports the potential of Malo jezero for future conservation and reintroduction efforts.

1. Introduction

The noble pen shell (Pinna nobilis L.) is a large bivalve species endemic to the Mediterranean Sea, found on sandy and muddy sediments and within seagrass ecosystems, particularly Posidonia oceanica and Cymodocea nodosa meadows, where it plays a critical ecological role that involves filtering water, stabilizing sediments, and providing a habitat for other organisms, which enhances biodiversity and habitat complexity [1,2,3]. This species is an integral part of these ecosystems and serves as a sentinel for environmental health, due to its sensitivity to habitat changes [4].

The population density of P. nobilis varies significantly across the Mediterranean, especially within lagoon environments, showing a wide range of densities and patchy distribution [5,6,7], and references therein]. In some regions, these densities are exceptionally high, indicating the capacity of some habitats to sustain dense populations. Some of the highest population densities were reported in Tunisia, 56 ind/100m² [5], and France, 70 ind/100m² [7].

Along the Mediterranean and Adriatic coast, P. nobilis has been documented to reach heights of up to 120 cm, making it one of the largest Mediterranean bivalves [8]. Individuals can live for over 20 years and exhibit rapid early growth, with subsequent slowing as they age [2,9]. Based on long-term monitoring data on their estimated total height, individuals aged 45–50 years have been reported [6].

Size (and age) population structure differ across the Mediterranean basin. Most populations show unimodal [2] or multimodal [10] size distributions, but some were reported to have bimodal distributions [3], indicating recruitment issues or environmental pressures, suggested by the absence of certain size and/or age categories. For example, surveys conducted in the Mar Menor lagoon between 2014 and 2019 revealed an absence of individuals smaller than 10 cm, suggesting recruitment failure or high predation during early life stages [11]. Populations in Sardinia were also found to be dominated by medium-sized individuals, with smaller size classes absent, pointing again to recruitment limitations [12].

The health status of P. nobilis populations depends on favorable environmental factors, such as water quality, substrate type, and recruitment rates; therefore, the average size distribution of this population typically includes a wide range of size classes, reflecting successful recruitment over multiple years [1]. Unfortunately, that is not always the case. P. nobilis populations have experienced sharp declines due to habitat loss, pollution, and illegal harvesting, over the last few decades [1,13]. Since 2016, mass mortality event (MMEs), most likely caused by Haplosporidium pinnae and Mycobacterium sp. infections, have further devastated populations, with mortality rates reaching up to 100% in some areas [14,15,16]. This has brought P. nobilis to the brink of extinction and resulted in its classification as Critically Endangered by the IUCN in 2019 [17].

While much research has focused on documenting these declines, less attention has been paid to the historical population structures of P. nobilis, particularly in enclosed and semi-enclosed environments. Such habitats are characterized by restricted water exchange with the open sea, resulting in unique environmental conditions that shape population dynamics [18]. Understanding the historical population structure of a species on the verge of extinction might be the key to tracking ecological trends and evaluating the resilience of P. nobilis populations to environmental change.

This study examines the historical population structure of P. nobilis in Malo jezero, a coastal lagoon within Mljet National Park, Croatia. Using data collected before the MME, we analyze the population’s density, size distribution, and age structure. The goal is to provide a baseline demographic profile for this distinctive ecosystem and to understand how the population was structured before its collapse. The results might help to assess the suitability of the lagoon as a conservation sanctuary, identify potential threats, and inform management authorities.

2. Materials and Methods

2.1. Study Location

The research was conducted during July and August 2018 in Malo jezero lagoon in Mljet National Park (Figure 1). Malo jezero is the smaller of two lagoons, formed in a karstic depression during the post-glacial period [19]. The bigger lagoon, Veliko jezero, is connected to the open sea through a 2.5 m deep and 10 m wide strait and to Malo jezero through a 0.5 m deep and 30 m long channel. The surface of Malo jezero is 0.24 km², with a maximum depth of 29.4 m. During warmer periods, the lake exhibits strong stratification, with a pronounced thermocline, leading to oxygen depletion in deeper layers [19]. The average monthly sea temperature measured at a depth of 80 cm during the survey period was 25.8 ± 0.2 °C in July and 27.6 ± 0.2 °C in August 2018. Veliko jezero and Malo jezero are no-take zones within the boundaries of Mljet National Park.

2.2. Sampling Design and Density Measurement

Due to the high density of P. nobilis individuals spread over the large area within the lagoon, a grid-based transect system was used. Eight identical grids covering a total area of 11,520 m² were used. Each grid covered an area of 48 m by 30 m (1440 m²) and consisted of six parallel strip transects. Each transect was 30 m long and 4 m wide on either side (240 m²). A rope of four meters tied to the central line was used to delimit the boundaries of the strip, following the protocol established in [20]. Using the grid, the avoidance of any individuals was reduced to a minimum. During the research, the grid was repositioned by a team of four SCUBA divers, so that at least two points of the square remained fixed at every reposition. The square points were georeferenced with the GPS Garmin GPSMAP 64x, Software version 4.20.

Surveys were conducted by trained SCUBA divers, operating in pairs, with each pair assigned to a transect. Divers recorded all visible P. nobilis individuals along the transect lines, measured the pinnid biometrics to the nearest centimeter (1 cm) using calipers, and recorded the position on the transect for spatial analysis. The P. nobilis population density was calculated for each transect by dividing the total number of individuals observed by the transect area (240 m²) and standardizing the results according to the number of individuals per 100 m².

The total abundance of Pinna nobilis in Malo jezero was estimated and modeled by multiplying the average population density (individuals per 100 m²) by the surface area of the lagoon within the depth range of 2–15 m. This depth range reflects the habitat suitability for P. nobilis, based on previous observations wherein the species had been recorded at depths of up to 15 m [2], and the recorded depths from this study (2–8 m). This expanded depth range was used to ensure comparability with earlier findings and to provide a comprehensive estimate of population abundance.

2.3. Size and Age Estimation

According to the established protocol [20], the height above the sediment (H_s), maximum width (W), and width (w) at the sediment level were measured. For the estimated total height (H_t) of individuals in Malo jezero lagoon, a formula from [21] was used:

H_t = 1.18W + 40.39.

Compared to other models, this simple linear regression provides an accurate prediction of the overall height of the individuals.

A growth curve for P. nobilis was generated using the von Bertalanffy growth equation:

H_t = H_t∞ (1 − e^−kt);

where H_t∞ is the value of the maximum theoretical total height for this population (72.31 cm), and k is the slope of the line (0.16), calculated for Adriatic populations of P. nobilis [2], and used here to ensure regional specificity.

Using this model and the size–age relationship established in 2003 [2], we estimated the ages of P. nobilis individuals based on their shell height. The equation defines a theoretical maximum age of 44.34 years, beyond which age estimates are unreliable. Individuals exceeding this range were excluded from the statistical analysis of age, but they were included in the histograms to provide a complete overview of the population structure.

2.4. Statistical Analysis

The data were organized and processed using MS Excel for Mac (Version 16.93.1). The statistical analyses were conducted using the R programming language (ver. 4.3.3, Angel Food Cake) [22] and R Studio software (ver. 2023.12.1, Ocean Storm) [23]. Statistical significance was assessed at an alpha level of 0.05.

The average depth data did not follow the normality assumptions (Shapiro–Wilk W = 0.92, p = 0.003), so Spearman’s rank correlation was used to assess the relationship between the average depth and the P. nobilis density for each transect.

Geospatial analyses, including creating heatmaps, geographical maps, and surface models, were performed using QGIS (ver. 3.22.5, Białowieża) [24]. All the data and protocols associated with this study are available upon request from the corresponding author.

This study complied with all the ethical guidelines and regulations for field research, and permissions were granted by the Ministry of the Sea, Transport, and Infrastructure in 2018.

3. Results

A total of 3800 Pinna nobilis individuals were recorded across 48 transects, each covering an area of 240 m². Among these, 483 individuals were dead, indicating a mortality rate of 13%. All further calculations were based solely on living individuals.

3.1. Density

The density values were standardized to the number of individuals per 100 m². Across the transects, the density ranged from 7.50 to 55.83 individuals per 100 m², with a mean of 25.42 individuals per 100 m² (SD = 11.83) and a median of 25.00 individuals per 100 m².

Higher densities were observed in transects located at greater depths (Figure 2), particularly in the central sections of Malo jezero (e.g., transect 7E, 55.83 individuals per 100 m²). Spearman correlation analysis confirmed a significant positive relationship between depth and density (rs = 0.70, p < 0.001, n = 48) (Figure 3). As shown in

Table 1. Transect-level data showing Pinna nobilis counts, densities (ind/100 m²), and average depths recorded in Malo jezero, Mljet National Park. Transect areas are standardized to 240 m².

Transect Code	Count (per 240 m2)	Density (ind/100 m2)	Average Depth (m)
1A	42	17.5	2.4
1B	33	13.75	2.6
1C	38	15.83	2.5
1D	24	10	2.9
1E	55	22.92	3.1
1F	39	16.25	3.2
2A	56	23.33	2.8
2B	65	27.08	3.0
2C	77	32.08	3.0
2D	87	36.25	3.0
2E	75	31.25	3.4
2F	61	25.42	4.0
3A	22	9.17	2.0
3B	46	19.17	2.0
3C	37	15.42	3.0
3D	40	16.67	3.5
3E	61	25.42	4.0
3F	45	18.75	4.0
4A	19	7.92	2.5
4B	18	7.5	2.5
4C	23	9.58	3.3
4D	28	11.67	3.7
4E	39	16.25	3.5
4F	37	15.42	5.5
5A	115	47.92	4.4
5B	122	50.83	4.2
5C	88	36.67	3.5
5D	97	40.42	3.2
5E	92	38.33	3.7
5F	74	30.83	2.5
6A	87	36.25	5.0
6B	88	36.67	5.4
6C	79	32.92	5.0
6D	66	27.5	5.0
6E	97	40.42	5.0
6F	91	37.92	4.5
7A	69	28.75	6.0
7B	104	43.33	6.2
7C	78	32.5	6.3
7D	93	38.75	7.0
7E	134	55.83	7.0
7F	91	37.92	7.2
8A	120	50	6.0
8B	87	36.25	5.2
8C	110	45.83	6.0
8D	105	43.75	7.0
8E	110	45.83	7.2
8F	104	43.33	7.0

, transects exceeding 5 m in depth consistently exhibited greater population densities than shallower areas. Figure 2 provides a heatmap visualization of the density distribution, highlighting the spatial variation observed across the study site.

Given the mean density of 25.42 individuals per 100 m² and the total surface area of Malo jezero within the modeled depth range of 2 to 15 m (0.123 km²), the total abundance of P. nobilis in this coastal lagoon at the time of the study was estimated to be approximately 31,266 individuals.

3.2. Size and Age Distribution

The measured shell widths ranged from 10.0 to 33.0 cm, with a mean of 20.41 cm (SD = 2.20) and a median of 20.0 cm (Table S1).

The estimated total shell height, calculated using the width-to-height equation [21], ranged from 52.19 to 79.33 cm, with a mean height of 64.48 cm (SD = 2.60) and a median of 63.99 cm (Table S1; Figure 4).

The estimated ages of P. nobilis individuals in Malo jezero were calculated using the size–age equation [2]. The resulting growth curve (Figure 5) illustrates the modeled relationship between shell height and age. It reveals rapid growth during the first decade of life, with growth rates slowing as individuals approach the maximum theoretical height. The population’s age ranged from 8.00 to 44.34 years. The mean estimated age was 14.29 years (SD = 2.98), and the median age was 13.51 years. Based on the size–age equation, 20 individuals exceeded the theoretical maximum shell height of 72.31 cm predicted by this growth model, with measured heights ranging from 73.43 cm to 79.33 cm. These exceptionally large individuals could not be aged accurately using the model and were likely to be older than 44.34 years.

Figure 6 provides a histogram of the age distribution for individuals included in the analysis, with outliers visually represented to highlight their distinctiveness within the population structure.

4. Discussion

This study provides a retrospective look at the historical population structure, size, and age distribution of the noble pen shell, Pinna nobilis, in Malo jezero, Mljet MPA. It offers a baseline for understanding its ecological characteristics and informing future conservation strategies.

4.1. Density

The mean density (25 ind/100 m² over a large area of 11,520 m²) recorded during this research is higher than previously published for Malo jezero [2,25] or the rest of the Adriatic Sea, according to which previous reports indicate a mean density of approximately 10 ind/100 m² [8]. In a systematic review from 2015 [26], 24 papers on population density were identified, and the average population density of 9.78 ind/100 m² in the Mediterranean was calculated. The authors noted high and statistically significant variability among different ecoregions. Moreover, they detected high variability in the methodology, which is important to consider given the species’ patchy distribution. The high mean abundance over a large area (and not only isolated patches), in the present study, indeed indicates an unusually high abundance of P. nobilis in Malo jezero, compared to other Mediterranean regions.

The observed positive correlation between depth and P. nobilis density (Figure 3) within the studied depth range (2–8 m) aligns with findings in previous works on Malo jezero. In 2002, the highest densities were found between a depth of 5 and 10 m [25], and high densities were reported in 2003, in Cymodocea nodosa meadows, at depths of 7–8 m [2]. It also corroborates the findings from Tunisia in 2010, where the density increased with depth, within the studied depth range (0–6 m) [5]. These patterns highlight the importance of habitat structure and depth as key drivers of P. nobilis distribution across the Mediterranean.

4.2. Size and Age Distribution

Given the mean total height of 64.48 cm, the results reveal the presence of many mature individuals, with no evidence of juveniles or recent recruitment (Figure 4). Moreover, as previously recorded in Malo jezero for the same population, an individual whose total height was as much as 84 cm was recorded [21]. This specimen was dead and, therefore, was not included in the current analysis.

The age range from 8.00 to 44.34 years indicates the absence of recent recruitment and reflects a population dominated by mature individuals. Similar patterns were observed in other Mediterranean locations, such as Port-Cros National Park, where an extreme case of a lack of recruitment for more than 40 years was observed [6]. Other authors have also reported the lack of juveniles in populations of P. nobilis [11,12]. Interestingly, in 1980, no P. nobilis individuals were recorded in Malo jezero [27], yet a dense population was documented in 1996, with juveniles observed in 1998 [28]. Later, juveniles were recorded in 1998 and 2000, and recruitment and growth patterns for P. nobilis in Malo jezero were documented [2]. However, between 2000 and 2018, there was no recruitment, and no juveniles were observed in the present study. The reasons for this lack of recruitment are unclear, but one possible explanation might be that the hydrodynamic isolation of the lagoon is a barrier to larval exchange with open populations [18]. Limited connectivity with the open sea restricts both horizontal and vertical water circulation, further reducing larval dispersal and exchange with external populations [29]. The prolonged larval retention time in Malo jezero, estimated to be 160–185 days, suggests that while some larvae may have settled within the lake, the restricted water exchange likely limited both genetic diversity and the influx of new recruits from external populations, further exacerbating the observed recruitment failure [18]. Another factor contributing to the existence of the aging population could be the threshold “refuge size”, where individuals above a certain size are less vulnerable to predation and environmental stressors and remain in the population when recruitment is absent [30].

The maximum estimated age of 44.34 years falls within the upper limit of the documented age for P. nobilis [6]. The presence of 20 exceptionally large and old individuals (>72.31 cm), for which the age could not be estimated, together with 13 individuals with a total height larger than 72 cm (older than 44 years), but within the model boundaries, accounts for approximately 1% of the total population that has reached beyond the theoretical maximal height. This is in contrast with research from 2020, which discovered that local hydrodynamism is usually lower in enclosed environments and supports populations in which the theoretical maximal height is higher than the observed maximal height [31]. The individuals recorded in our study could represent a subset of the population, with growth characteristics not accounted for in previous models (Table S1; Figure 6). This result underscores the uniqueness of our population and highlights the potential for variability in growth in specific environmental conditions.

Following the MME that began in 2016, no living P. nobilis individuals have been observed in Malo jezero. Surveys conducted after 2019 have confirmed 100% mortality, leaving only empty shells, mirroring the widespread collapse of the species across the Mediterranean [14,15,17]. The enclosed nature of Malo jezero, which historically may have contributed to high population densities, likely became a disadvantage following the MME, as limited water exchange restricts larval dispersal and natural recolonization. Similar challenges have been documented in other enclosed environments, such as the Mar Menor lagoon [11], reinforcing the need for targeted conservation strategies. The pre-MME data serve as a crucial reference for potential reintroduction and restoration efforts, which can aid in understanding population dynamics and support efforts to develop strategies for reintroduction and restocking [32].

5. Conclusions

This study provides a rare insight into a now-extinct population, highlighting both the past ecological success and the fragility of P. nobilis in the face of widespread environmental threats. Many individuals, a high population density, and the lack of juveniles or smaller individuals observed in Malo jezero before the MME highlight the vulnerability of P. nobilis populations when there is no recruitment. Populations consisting mostly of old individuals are especially at risk from sudden events, as they lack the younger generations needed to recover and sustain the population. The enclosed nature of the lake, which may have historically supported high population densities, ultimately became a barrier to recolonization following the collapse of the population.

Our study offers valuable insights into the historical population structure of P. nobilis. Based on the legal protection of the site (a no-take zone within the Mljet National Park) and the sizes and ages reached, Malo jezero could be a favorable location for potential reintroduction efforts in the future.

The lessons learned from Malo jezero can inform conservation efforts aimed at safeguarding any remaining populations and, potentially, restoring P. nobilis to its former habitats.