The Marine Macroalgae Collection from Herbarium João de Carvalho e Vasconcellos (LISI)—140 Years of History

Abstract

Herbaria phycological collections have approximately one million 700 thousand specimens preserved in European herbaria, a significantly lower number when compared to vascular plants, due to factors such as greater sampling difficulty and fewer specialists. Several studies report that coastal systems have undergone dramatic ecological changes in the last 150 years, with macroalgae being a particularly affected group. Thus, macroalgal herbaria are essential sources for the study and conservation of this biodiversity, as well as a pillar that responds to several ecological questions. Despite having a large coastline, Portugal’s phycological collections are scarce, poorly developed, and practically inaccessible digitally. In 2021/2022, all the phycological specimens present at LISI were the focus of this exploratory project whose objective was to catalog them, taxonomically review the specimens and place them at the service of the scientific community through the incorporation of digitized vouchers into online databases. Three marine collections were constituted and studied, accounting for a total of 852 vouchers and more than 1800 specimens, being the Portuguese Marine Macroalgae Collection, the oldest digitized phycological collection available in Portugal. This project provides an opportunity for other educational institutions to embrace their long-neglected collections as well.

1. Introduction

After several decades of obscurity and neglect, Natural History Collections (NHCs) are now receiving a renewed interest, being appointed as valuable resources to ample and pioneering ecological studies [1]. In fact, herbaria remain the major source for both the discovery and subsequent formal description of plant taxa new to science, with more than 50% of yet-to-be described species estimated to already be in herbaria awaiting description [2,3].

Overloaded herbaria with unprocessed and unavailable species for study and a lack of expertise in particular taxa are some of the several reasons why older specimens representing new species remain undetected and undescribed in herbaria [2]. Additionally, and even with this renewed interest, the lack of voucher deposition by researchers may lead to weaker collections with vast geographical, temporal, or taxonomic gaps in their coverage and representativeness [1].

The first studies and the establishment of phycological collections for scientific purposes in Portugal began in the late 18th century with Vandelli [4] and the work of Correa da Serra [5]. In the following years, Welwitsch [6] and Hauck F. [7] also made contributions to this field.

The primary references to Portuguese marine algae correspond to the publications of Palminha [8,9,10,11], Mesquita Rodrigues [12,13], Póvoa dos Reis [14], and Ardré F. [15], with the latter representing the most comprehensive study of the marine algal flora in Portugal for many years. Ardré studied, identified, and described 404 species of macroalgae, including 246 Rhodophyta, 98 Heterokontophyta, and 60 Chlorophyta.

In 2009, Araújo et al. [16], based on the literature, new records, and herbarium data, compiled an updated survey of benthic marine algae along the northern coast of Portugal, resulting in 346 species (200 Rhodophyta, 70 Heterokontophyta, 50 Chlorophyta, and 26 Cyanobacteria). Among these, 33 species were recorded for the first time in this region, and 21 were recorded for the first time along the Portuguese coast. In recent years, new surveys have been conducted by other authors, including Pereira [17], Bárbara et al. [18,19], Díaz-Tapia et al. [20,21], and Pereira et al. [22].

Despite Portugal having several herbaria institutions with significant algal collections—including the National Museum of Natural History and Science, Faculty of Sciences, University of Lisbon (Herbarium LISU); the National Institute for Agricultural and Veterinary Research (INIAV) (Herbarium LISE); the Faculty of Sciences and Technology, University of Coimbra (Herbarium COI); the Faculty of Sciences and Technology, University of Algarve (Algarium ALGU); the Natural History and Science Museum, Oporto University (Herbarium PO); the Funchal Natural History Museum (Herbarium MADM); and the Faculty of Sciences and Technology, University of the Azores (Herbarium Ruy Telles Palhinha AZB)—most of these collections, especially the historical ones, are neither extensively studied nor readily accessible to the scientific community. Furthermore, none of these institutions, as far as the authors know based on direct contact with their researchers or via online information, have both digitized their specimens and added data to centralize information in the Global Biodiversity Information Facility (GBIF).

Therefore, this first project regarding the LISI phycological collection in the 21st century is focused on the Portuguese Mainland Marine Macroalgae and two smaller sub-collections, aiming to (1) organize the collection from an historical perspective and formally register it in the LISI database; (2) taxonomically update specimens already present; (3) incorporate new vouchers with associated accurate location information according to a geographic information system (GIS); (4) complement specimen identification with DNA barcoding methodologies; and (5) share DNA barcode sequences and voucher information with public repositories such as the Barcode of Life Data System (BOLD) and GBIF, contributing to populating these repositories with DNA barcode standards and associated digitized voucher information (both databased and imaged). The overall objective is then to enhance the accessibility of phycological collections in Portugal, particularly those containing historical specimens, while simultaneously increasing the volume of available data for study in this field.

Context of the LISI Herbarium

Instituto Superior de Agronomia (ISA) is the University of Lisbon’s School of Agriculture, which was founded in 1910. From the moment of its foundation, a general herbarium (LISI) was started, which, by 1917/1918, comprised about 3800 vascular spontaneous and 658 ornamental Portuguese plant specimens [23]. Among the list of illustrious LISI Herbarium curators are the names of António Xavier Pereira Coutinho and João do Amaral Franco. Under their wise leadership, the institution’s NHC grew steadily, reaching 60,000 specimens by 1986 [23]. Today, it holds more than 80,000 pressed and dried specimens, including 48 type specimens [24].

Currently, the herbarium holds five distinct vascular plant collections: (1) spontaneous vascular plants of the Iberian Peninsula (LISI-IBER), encompassing about 67,000 specimens (98% databased, 11% imaged); (2) spontaneous vascular plants of the Azores Archipelago (LISI-AZO), containing about 5300 specimens (100% databased, no specimen imaged); (3) spontaneous vascular plants of the Madeira Archipelago (LISI-MDR), having about 500 specimens (95% databased, no specimen imaged); (4) cultivated plants in Portugal (LISI-CULT), containing about 12,700 specimens (neither databased nor imaged); and finally a small collection of (5) world vascular plants (LISI-WLD), comprising about 6900 specimens (also neither databased nor imaged). Small collections of bryophytes and lichens and a carpological collection are also worth mentioning, although their exact size is not known at present.

Besides the vascular plants collection, LISI has a phycological collection, which earned little to no attention and was overlooked for a long period. This collection began with contributions dating back to the 19th century, before the institute’s foundation. Later, other collections were aggregated from various defunct scientific institutions. Recent additions to the collection have originated from several research projects as well as from casual, unplanned sampling. Due to the extensive collection of algal specimens, documentation, and other phycological materials available at LISI, it was decided in January 2021 to formally manage this historical collection by establishing a new herbarium collection named “Algarium LISI” (LISI-ALG).

2. Materials and Methods

2.1. Collectors’ Biographic Research and Historical Background

Following compiling all the material related to mainland Portugal’s marine macroalgae, the LISI collectors’ names and biographies were traced to identify the collection’s history as well as to understand the collection increment throughout the school’s different periods. Finding all existing documentation associated with this collection would also increase understanding of it, namely its sampling methodology and policy. The performed historical background research was mainly obtained via interviews during 2021 and via reports from Cândido Pinto Ricardo, João Monjardino, Lisete Caixinhas, and Teresa Vasconcellos. These researchers contributed during their careers in the 1960s, as well as contributing findings in paper publications related to the different sub-collections in the LISI archives.

2.2. Historical Specimens’ Taxonomic Revision

Considering the previously existing material, a relatively high percentage of the material was unidentified, had outdated taxonomic ranks (that is, with no meaning or synonym to today’s taxonomy), or had incomplete information regarding the taxonomic status and/or collection site.

In a first stage, taxonomic revision and identification began by using reliable identification guides and keys for morphological and histological identification for Portugal or the northern Atlantic regions, such as Pereira [17], or Hiscock [25,26] and Dickison [27], respectively. The use of the latter classical literature was employed not only to understand the taxonomic nomenclature present in historical specimens, but also to serve as a basis for using updated taxonomic resources. During recent years, numerous changes in species taxonomic status have been implemented due to the incorporation of molecular methodologies, with updates made available through the public repository AlgaeBase (www.algaebase.org; accessed on 26 April 2021. Consequently, the work of Guiry and Guiry [28] has been instrumental in determining the latest accepted taxon names for various specimens, including those collected in 2020 and 2021. For several genera, due to different reasons such as lack of information on the voucher and/or bad conservation status of the material, specific literature had to be analyzed to ensure a correct identification.

2.3. Geographic Coverage

Before the start of the current digitization project, the most recent vouchers dated back to 1983. Consequently, a new field sampling campaign was mandatory to update the collection and minimize the temporal gap. Samples were collected during the summer of 2020 and 2021.

Sixteen zones of rocky coast exposed to tides in mainland Portugal were selected, representing the sampling conducted between 1881 and 1983, to revisit and collect new material. The localities along the continental Portuguese coast, from North to South, are as follows: Praia da Apúlia (Esposende); Praia dos Beijinhos (Póvoa do Varzim); Praia Azul de Santa Cruz (Torres Vedras); Praia do Magoito (Sintra); Cabo Raso (Cascais); Cascais and Estoril (Cascais); Praia de Carcavelos, Parede, and Avencas (Cascais); Praia do Serro da Águia (Sines); Praia da Nossa Senhora (Odemira); Praia Grande (Odemira); Porto de Pesca de Lapa de Pombas (Odemira); Praia da Balaia (Albufeira); Praia da Rocha Baixinha (Albufeira); and Farol da Barra Nova (Faro) (Figure 1,

Table 1. Collection sites in 2020 and 2021, from 1 to 16 (north to south), and respective geographic coordinates.

Collection Sites	Sampling Date	Municipality	Latitude	Longitude
Praia da Apúlia	28 September 2021	Esposende	41.48454	−8.77985
Praia dos Beijinhos	28 Spetmeber 2021	Póvoa do Varzim	41.38846	−8.77764
Praia Azul e Santa Cruz	24 August 2021	Torres Vedras	39.11579	−9.393531
Praia do Magoito	12 August 2021	Sintra	38.85507	−9.454876
Cabo Raso	11 August 2021	Cascais	38.70885	−9.486002
Cascais e Estoril	18 July 2021	Cascais	38.70258	−9.407510
Praia das Avencas	27 July 2021	Cascais	38.68851	−9.36179
Praia da Parede	27 July 2021	Cascais	38.68446	−9.351555
Praia de Carcavelos	9 October 2021	Cascais	38.68059	−9.344986
Praia do Serro da Águia	10 Spetember 2021	Sines	37.86305	−8.79412
Praia da Nossa Senhora	10 Spetember 2021	Odemira	37.53103	−8.78692
Praia Grande	10 Spetember 2021	Odemira	37.64924	−8.804494
Porto de Pesca de Lapa de Pombas	10 Spetember 2021	Odemira	37.63587	−8.80977
Praia da Balaia	16 September 2021	Albufeira	37.08796	−8.211207
Praia da Rocha Baixinha	18 August 2021	Albufeira	37.07869	−8.143828
Farol da Barra Nova	4 March 2020	Faro	37.01498	−8.005066

).

2.4. In Situ Sampling Methodology

At each location, the rocky coastal section is accessible by foot, ranging from the upper intertidal zone or upper littoral fringe to the lower intertidal zone or lower littoral fringe, which was thoroughly surveyed by a team of two or three individuals over a two-hour period (one hour before low tide until one hour after low tide). The field trip dates were selected based on lunar phase, with a preference for weeks around the full moon or new moon when low tides are more pronounced.

With the assistance of pocketknives or blades, specimens were collected; cleaned of debris, sand, and animal organisms on-site using seawater; and placed in plastic containers without water for the remainder of the sampling process. The specimens were placed within the same container until a reasonable fill was achieved without material compaction. Samples of specimens intended for molecular studies were collected immediately, placed in small non-woven textile (TNT) bags similar to tea bags, cinched closed, and stored in a container with silica gel (high-purity grade, Davisil Grade 12, pore size 22 Å, 28–200 mesh), properly sealed to rapidly extract the sample’s water content and prevent material degradation. During the return journey to the laboratory, all sealed containers were kept in a dark and cool place to prevent material degradation.

2.5. Identification of Specimens Collected in Field Expeditions

The specimens collected in 2020 and 2021 initially underwent the same process of morphological and histological identification as the historical specimens. This process involved utilizing identification keys for morphological and histological characteristics specific to Portugal, the Iberian Peninsula, or the North Atlantic region. Some examples of referenced literature are Pereira [17,22,29] and Guiry and Guiry [28]. The specimens were observed through a magnifying glass, and optical microscopy preparations were made for observation.

2.6. Pressing and Specimens’ Preservation

All specimens collected during the sampling expeditions were preserved immediately after their morphological identification, following different criteria. Laminar specimens were soaked in containers of freshwater for rehydration. Subsequently, a sheet of watercolor paper was also soaked in the container and passed beneath the specimen. Carefully, the sheet would lift the specimen, fixing it in place. Using a brush, tweezers, and scalpel, the specimens were meticulously groomed, meaning they were prepared to display their morphological characteristics while removing excess material. They were then covered with sheets of culinary parchment paper and placed between herbarium sheets for pressing.

The herbarium sheets were changed after a few hours and again 24 h later. The pressing and drying process varied depending on the specimens, usually taking around 72 h. Larger specimens were accommodated on A3- or A4-sized sheets, while diminutive specimens were placed in small envelopes. Due to their morphology and attachment to substrates, coralline specimens were stored in plastic containers having transparent lids.

2.7. Preservation and Storage Methods in the Herbarium Room

After the completion of identification studies, computerization, and digitization, it was necessary to subject the herbarium specimens to preservation and conservation processes to eliminate potential infesting beetle eggs and other harmful organisms like fungi, before they were stored in the Herbarium Room in their new space. For this purpose, all specimens were placed in sealed plastic bags and introduced into a refrigeration chamber at a temperature of −18 °C for seven days. Subsequently, the material was transferred to an individualized and specific cabinet designed to store these studied specimens in the Herbarium of Ornamental Vascular Plants, Carpological Collection, and Lichens Room at a temperature of 16 °C and 58% humidity.

2.8. Data Treatment

All the information contained on each specimen’s identification label was entered into a database containing details about the specimen collectors, date, location, habitat, preparations, owning collection, preservation method, determinations, and other relevant observations.

Before they could be digitized, a label with the new herbarium number and a QR code was attached to each herbarium sheet, envelope, or box—depending on the preservation method of the material. Subsequently, the I2S eScan Open System Scanner (planetary scanner) was used for capturing images of specimens for the Collection, using LimbCapture software version 1.10.66, following the internal protocol.

After all the specimens were digitized, a verification and matching process of all information present in the database with each of the specimens was performed to make the dataset available on the GBIF platform.

3. Results

After assembling and sorting all algal material available at LISI, the collection was organized according to Figure 2. Four sub-collections were, thus, created: (1) Portuguese Mainland Marine Macroalgae; (2) Azores Archipelago Marine Macroalgae, collected by Ilídio Botelho Gonçalves; (3) Foreign Marine Macroalgae, encompassing Welsh Marine Macroalgae, collected by Cândido Pinto Ricardo; the Farlow Herbarium (FH)—Harvard University duplicate vouchers stack; and French Marine Macroalgae, collected by Emile Deyrolle; and lastly, (4) Freshwater Macroalgae, where we locate “Rice Paddies Invasive Macroalgae”, a research project led by João de Carvalho e Vasconcellos during the 1950s; and some accessory documentation on the projects that led to this material. The global collection also includes associated documents providing little scientific or historical information.

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Portuguese Mainland Marine Macroalgae collectors’ history

Overall, it is possible to group the LISI collectors in two main clusters: members of Sociedade Broteriana, the oldest Portuguese botanical non-governmental organization, founded by botanic professor Júlio Henriques from Coimbra University in 1880; and researchers or technicians somewhat related to ISA or one of the pre-existing institutions linked to the foundation event of ISA (e.g., Instituto Geral de Agricultura and Instituto de Agronomia e Veterinária). The total number of collectors amounts to 30, and their names are summarized in

Table 2. Historical LISI collectors, their collector period regarding this collection, and their affiliation to the Institute. (*) old designation for Instituto Superior de Agronomia; (**) old designation for Instituto Nacional de Investigação Agrária e Veterinária.

Name	Collector Period	Affiliation
A. D. Moreira Padrão	1883	Sociedade Broteriana
A. Goltz de Carvalho	1883–1890	Sociedade Broteriana
Cândido Pinto Ricardo	1962–1985	Instituto Superior de Agronomia
Carlos Bobone	1938	Sociedade Broteriana
Carlos Galrão	1885–1886	Sociedade Broteriana
Conde de Bobone	Late 1890s	Sociedade Broteriana
Cunha e Sousa	1940	Instituto Superior de Agronomia
D. Sophia Rosa da Silva	1881–1886	Sociedade Broteriana
F. Nascimento	1982	Instituto Superior de Agronomia
J. Batalha Reis	1870s	Instituto Geral de Agricultura *
J. G. de Barros e Cunha	1890	Sociedade Broteriana
J. P. Simões	1982	Instituto Superior de Agronomia
João de Mendonça	1880–1884	Sociedade Broteriana
João Rafael Monjardino	1967	Instituto Superior de Agronomia
José E. Mendes Ferrão	1964	Instituto Superior de Agronomia
José Gomes Pedro	1982	Instituto Superior de Agronomia
Luís (Filipe Lopes) Martins	1966–1967	Unknown
Luiz Martins	1947–1950	Unknown
M. Reis Moreira	1900s	Unknown
Maria Alice Guedes Pinto	1957	Instituto Superior de Agronomia
Maria Helena Pereira Dias	1965	Instituto Superior de Agronomia
Maria Lisete Caixinhas	1966–1967	Instituto Superior de Agronomia
Maria Micaela da Fonseca	1961	Instituto Superior de Agronomia
Mário Myre	1940	Estação Agronómica Nacional **
Marques de Sousa	1940	Estação Agronómica Nacional **
Miguel Pereira Coutinho	1967	Instituto Superior de Agronomia
Nestor Mendes	1940	Estação Agronómica Nacional **
Rafael Mafra	1979	Estação Agronómica Nacional **
Silva Pinto	1940	Estação Agronómica Nacional **
Teresa Isabel E. Sezinando	1977	Instituto Superior de Agronomia

.

3.1. Temporal Coverage

The sub-collection Portuguese Mainland Marine Macroalgae currently has a total of 737 specimens. Of these, 77 have no collection data. The sub-collection began in 1880, and the last historical examples date from the 1980s. After a 40-year gap, in 2020 and 2021, 292 new specimens were presented in the sub-collection, generating an increase of 44% of the collection known to date (Figure 3 and Figure 4).

The oldest voucher present in this collection is a red algae (Rhodophyta) specimen Ellisolandia elongata (J.Ellis & Solander) K.R.Hind & G.W.Saunders, collected by João de Mendonça in September 1880 at Baía de S. Martinho (São Martinho do Porto, Leiria district) (Figure 5a).

3.2. Taxonomic Coverage

The Portuguese Mainland Marine Macroalgae collection has 116 specimens belonging to the division Chlorophyta, 217 to the division Heterokontophyta, and 408 to the division Rhodophyta (Figure 6), distributed along 51 families (Figure 7).

3.3. Geographical Coverage

The geographical distribution by district of the specimens of the collection demonstrates that the largest number of specimens collected occurred in the district of Lisbon, with 369 specimens (Figure 8.).

3.4. Seasonal Coverage

The seasonal distribution of the collection shows that the months with the highest number of collections are July, August, and September, with 79, 220, and 172 specimens collected, respectively. There are 131 specimens without any indication of their collection month (Figure 9).

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The sub-collection of French Marine Macroalgae by Emile Deyrolle

This collection was built by Emile Deyrolle (1838–1917), who became responsible in 1866 for the family house of the Deyrolle family, founded in 1831 by his grandfather Jean-Baptiste Deyrolle, at a time when natural history was gaining fame and numerous entomological societies were established throughout Europe. This institution extended its teaching and pedagogical aspects to about 120 countries by providing educational materials for schools, colleges, technical centers, and scientific institutions. Among the most popular scientific materials, pressed macroalgal specimens could be found [30].

In total, 64 specimens from Bretagne (Brittany) were found, with the oldest specimens dating back to 1885 and the most recent to 1888. Among this set of specimens, there are two whose date is not known. The sub-collection is composed of 5 specimens from the division Chlorophyta, 9 specimens from the division Heterokontophyta, and 50 specimens from the division Rhodophyta, encompassing 21 families. The most represented are Wrangeliaceae with 10 specimens; Callithamniaceae with 9 specimens; Gelidiaceae and Sargassaceae with 5 specimens each.

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The sub-collection of Welsh Marine Macroalgae by Cândido Pinto Ricardo

This sub-collection stems from the collections made by Professor Cândido Pinto Ricardo on the Isle of Anglesey, Wales, during a phycology course while preparing for his doctoral thesis in 1966. All the specimens belonging to this sub-collection were gathered in the month of March; due to their being collected in a specific location and time frame, it was determined that they would be considered a small sub-collection.

It is not possible to estimate the age of the oldest specimen in this sub-collection, as all specimens were collected within the same period.

The sub-collection comprises 3 specimens belonging to the division Chlorophyta, 14 specimens belonging to the division Heterokontophyta, and 32 specimens belonging to the division Rhodophyta distributed among 23 families. The most represented families are Fucaceae and Wrangeliaceae with 6 specimens each, and Rhodomelaceae with 4 specimens.

3.5. Data Sharing

The dataset with the data obtained during this project can be consulted at this link: https://www.gbif.org/pt/occurrence/gallery?dataset_key=57263ed3-c708-4523-92a4-d47e10381b53 (accessed on 26 December 2022) [31]

4. Discussion

Few tasks are more challenging than using a morphological key and other primary sources of the literature to identify marine macroalgae [32]. This difficulty arises from their simple morphology and anatomy, evolutionary convergence, remarkable phenotypic plasticity in response to environmental factors, and a lack of understanding of their life cycles with heteromorphic generations [33,34]. Additionally, phenology is a dominant and often overlooked aspect in plant ecology, whether at an individual or ecosystem level [35]. This gap is particularly relevant in photosynthetic organisms that are highly attuned to their environment, especially in an era of climate change where temperatures influence development periods, either independently or through other environmental cues, such as photoperiod [36].

Therefore, despite their usefulness, existing identification keys do not address these issues and are effective only at specific stages of the life cycle or for certain genera, rendering many organisms unidentifiable. Furthermore, their use often requires a high level of expertise that still does not eliminate the possibility of misinterpretation [37].

Another point to highlight, adding to the inherent difficulty of purely morphological and histological identification, is the significant taxonomic and phylogenetic rearrangement of marine macroalgae due to the use of molecular methodologies. Thus, there are now several taxa in which an accepted identification within the expert community can only be achieved through DNA sequence data; in others, achieving proper identification requires morphological and histological identification combined with highly specialized literature and molecular methods. For the red algae, two well-known examples are the genera Polysiphonia and Laurencia, which have both been split into numerous genera. These nomenclatural difficulties raise questions about how species are defined, as many taxa are classified based on morphological, molecular, or mixed characteristics [38].

Beyond the biological challenges, human-caused issues also come into play due to the impact of time on the various specimens. The current collection, containing specimens dating back to the late 19th century, has not, due to various circumstances, been properly conserved throughout its existence. This lack of maintenance, combined with the natural degradation of the organisms, resulted in a high percentage of advanced deterioration, initially hindering rapid and accurate identification. A considerable percentage of specimens found in this collection would, for many researchers in similar projects, be discarded due to lacking collection date and/or location or due to being significantly degraded, making correct identification impossible within the implemented protocol. However, this was not the approach taken during this project. All the existing historical specimens in LISI were valued and included in the collection. This choice resulted in additional efforts to recognize handwriting, herbarium paper types, and other unwritten information that allowed them to be categorized, with a level of uncertainty no greater than that for fully identified specimens, within the collection.

Overall, several taxonomic groups were identified with a high degree of certainty only to the taxonomic level of genus through practice and daily study in the identification of many specimens of each taxon.

NHCs have been constructed, curated, expanded, and used for various reasons over time, evolving significantly from being mere curiosities to becoming true repositories of ex situ biodiversity [1]. Traditionally, a classic collection is classified as a collection built and studied by botanists in the 17th, 18th, or early 19th centuries [39].

However, in the early 21st century, considering the dramatic historical events of the 20th century and the subsequent unprecedented social and environmental transformations, even collections built during World War II can be considered from this perspective as “classic collections” [39]. Thus, we can consider this current collection—which encompasses all the sub-collections studied in this project and whose earliest known specimen dates to 1800—a classic one and potentially representative, if not of various periods of research in marine botany, at least of some taxa within mainland Portugal.

Many questions remain unanswered regarding the study of this collection. It is important to note that due to temporal constraints, this project could not study the full collection. Therefore, many unanswered questions are related to this limitation.

Firstly, it is imperative to focus on the uncertainty of representativeness for comparative biodiversity studies of the general collection and the various sub-collections studied. This tentative lack of representativeness stems from two main factors. The first is the lack of knowledge about all specimens in this general collection of marine macroalgae, estimated to be at least double the number identified to date. Thus, the ALG-LISI collection is estimated to exceed more than 2000 specimens of just Portuguese mainland marine macroalgae. A complete understanding of the entire collection would allow us to assert the degree of representativeness of this phycological flora heritage with more data and certainty.

The second factor arises from a decline in interest in the discipline of botany since the late 20th century and a decrease in the number of specialists, effectively erasing a period of great taxonomic activity for the flora in the early 20th century [40]. This collection has also suffered the consequences of this decline, as observed in the results to date, showing a hiatus of 40 years in specimen collection. This temporal gap is primarily due to a paradigm shift in science. This declining trend continues to this day, becoming more pronounced in academia, resulting from factors such as funding cuts that have consequently reduced human resources in natural history museums and herbarium research units [41] or the methodological reductionism in biology, largely due to the extraordinary advances in technology and molecular biology, unintentionally leading to the devaluation of botany [42].

This lack of interest in the discipline, already detrimental on its own, coupled with a lack of information about the experimental designs used by various collectors throughout history, prevents us from understanding the extent, rigor, and intensity of the collections. For some authors [43], increasing scientific communication about exciting advances in plant knowledge and their importance to society could be a strategy to counteract the decline of botany. According to them, recent trends in public perception of plants are being explored using technological applications.

For many years, the use of NHCs for studies of biological representativeness has been criticized for limitations such as the absence of sampling strategy documentation and differences in experimental design over time, resulting mostly in ad hoc sampling [44], among other issues. In this regard, analyzing the different graphs related to both temporal increment and seasonality of collections in this collection, one can observe that the most sampled months are July, August, and September. This trend encompasses three interconnected reasons: (1) the summer period in Portugal coincides with the vacation periods of various collectors, facilitating easy collection; (2) this time of year is ideal for fieldwork due to favorable logistical conditions, contrasting with the winter season when rough sea conditions rarely allow safe work on rocky shores; and (3) organisms, in their most characteristic morphology in our climate, often coincide with the appearance of diagnostic blades and reproductive structures, as the richness and diversity of communities in continental Portugal are influenced by coastal upwelling regimes, which occur on the west coast and peak during the summer months [45].

Regarding taxonomic biodiversity, the studied collection presents more than half of its specimens from the division Rhodophyta. This fact is explained by this division having many marine macroalgal species, approximately between 6500 and 14,000 estimated species [38,46]. Next, there is a percentage of Heterokontophyta (between 15 and ~30%), which is generally twice the percentage of Chlorophyta in each sub-collection. Within the Chlorophyta division, only 10% of the species are marine [38]. However, Michael Guiry [38], one of the foremost macroalgal specialists, estimates that the Heterokontophyta division has 21,000 species, with around 11,600 species already described on his platform. This lesser representativeness can be explained by most of these species requiring a stable and rigid substrate to attach to [47], occurring mainly in rocky areas at depth and/or in areas unsuitable for beach activities in mainland Portugal, contrasting with the sandy shores with few rocks where many collections were made. Additionally, there are a significant number of filamentous brown macroalgae of small dimensions and epiphytes on others [47], which may have gone unnoticed during collection and identification or may have even been considered debris or dirt and removed from their host’s blades during the pressing process. In contrast, in this project, during the pressing process, various epiphytic specimens were kept together with their hosts to preserve relationships between distinct species in a marine context.

Among the 51 families, the most represented taxonomic groups are Ulvaceae, Sargassaceae, Gelidiaceae, and Fucaceae, in decreasing order of abundance, reflecting the occasional nature of this sub-collection’s sampling and the lack of specialists among the collectors. The Ulvaceae family encompasses a group of the most ubiquitous marine organisms, benefiting from eutrophic environments and those altered by anthropogenic impacts due to their opportunistic lifestyle [48]. The Sargassaceae, Gelidiaceae, and Fucaceae families have a strong ethnographic presence in our country. The first family is linked to the tradition of Sargaceiros; the second is used for agar production, having various applications; and the third is known for the popular tradition of “popping” the “bladders” (aerocysts), which gives it one of its common names. Thus, all these families, being easily identifiable by a broad array of collectors and with abundant distribution along the entire continental coast, are represented by many specimens and duplicates in the Portuguese Mainland Marine Macroalgae sub-collection.

In terms of the geographical distribution of the sub-collections, this collection reflects the characteristic socio-economic context of each period in its history. Most specimens were collected in the Lisbon district, explained by the working and personal relationships of the various collaborators with ISA. Many of the sampled locations in the city no longer exist or are completely degraded, such as Belém Beach or Cacilhas Beach. These specimens would, for example, allow us to understand the transformations and artificialization of the Lisbon coastline and particularly to make inferences about the loss of biodiversity in these human-transformed places. The specimens collected by members of the Sociedade Broteriana clearly show a consistent trend of being the result of expeditions in the Coimbra and São Pedro de Moel area, a region to which many of the members belonged, as this Society was founded there. As far as is known, there are few specimens that have reached ISA, and many of them are possibly duplicates of others that are likely in the University of Coimbra Herbarium.

Thus, with the data available to date, using this NHC to infer temporal changes in biodiversity, by using biodiversity indices in locations sampled in the past and during this project, would be fruitless and scientifically incorrect due to a lack of information. This statement can be understood based on the previously conducted analysis, which clearly demonstrates that the sampling effort is not focused on specific time periods but has been gradual over the past 140 years, with the known exceptions at the time being the collections made by Lisete Caixinhas, at the request of João de Carvalho and Vasconcellos, with a clear goal of increasing this particular collection during the 1960s, and the present project, which increased the known general collection by about 44%. Thus, the next step would be to study the remaining specimens of the collection and, based on the new results, analyze which scientific questions can be answered.

5. Conclusions

This project highlights the pivotal importance of herbaria as repositories of biodiversity and their potential for studies in phylogeny, biogeography, evolutionary adaptation, and genomics, among others. Recent but rare efforts to digitize macroalgal collections have contributed to expanding the audience benefiting from this scientific information. However, if they are to continue serving as a significant resource for future scientific generations, funds and other resources are essential. In the last decade, scientific journals have adopted policies to collect data to make them publicly available. However, database policies rarely, if ever, include requirements for linking to physical specimens (vouchers) stored in NHCs [41]. This has been the case with the GenBank database, despite recommendations in that direction. On the other hand, current collections are notably rich in rare species but often exhibit a significant scarcity of common species [1]. While the exclusive use of data resulting from some of these collections is not advised for some cases of inference in comparative studies due to poor or misunderstood sampling, there are several other studies that can be undertaken. One suggested type of study is the examination of marine flora responses to anthropogenic changes, and the currently constituted collection will be valuable for comparative purposes in the future. In this case, according to Johnson et al. [49], collections with many common taxa, are considered more useful as “time series” for determining species-level responses. Last but not least, this research has provided a substantial set of information data in the field of phycology and should be used as a case study for the reassessment of historical and musicological collections, especially the often-neglected phycological collections, in the digital era.