Feasibility of Biomarker-Based Taxonomic Classification: A Case Study of the Marine Red Alga Laurencia snackeyi (Weber Bosse) M.

: Taxonomy—the classification of species—is an important branch of biology that allows us to systematically understand and study biodiversity. Conventional taxonomy relies heavily on morphological and anatomical structures for classification, but recent discoveries of potentially cryptic species and morphological plasticity in many species underscore the importance of having an alternative or complementary method for species classification. In this paper, we discuss the emerging method of classification using biochemical signals, i.e., chemotaxonomy. We also present a case study on the feasibility of biomarker-based chemotaxonomy on the marine red alga Laurencia snackeyi using halogenated snyderane-type sesquiterpenes, which were proposed in earlier studies.


Introduction
Morphological classification, anatomical classification, molecular classification, and biochemical classification are some of the taxonomic approaches developed and used for studying and classifying organisms.The first two approaches are known as traditional taxonomic classification methods, while the other two are known as modern classification methods [1].Because morphological and anatomical characteristics alone are insufficient for taxonomy and phylogeny, complementing such data with modern classification methods is of significant value in the proper delineation of taxa [2].In this paper, we argue that biochemical classification (chemotaxonomy), like molecular classification, is a useful tool for classifying organisms based on the differences and similarities of their biochemical compound composition via metabolite profiling [3].
Based on PubMed records, the term "chemotaxonomy" was first mentioned in a paper by Hegnauer in 1958 [4].However, the number of publications on this topic only began to increase rapidly since 1998.The emergence of chemotaxonomy has provided significant insights into taxonomic classification, particularly in classifying those groups of organisms that cannot be differentiated from others or classified into specific taxonomic units based on their morphological and/or physiological characteristics, such as microbes and marine invertebrates [5].Most microorganisms, including bacteria, fungi, and microalgae, have simple and sometimes similar morphological structures, making it difficult to differentiate between taxa using morphological characteristics even when observing them under a microscope.Subsequently, recent taxonomical and biodiversity studies have employed polyphasic approaches, which use morphological characteristics complemented by molecular or biochemical data for species classification [6].
Phenotypic or morphological plasticity in response to environmental changes is another common factor that contributes to uncertainties in classification [7,8].This phenomenon affects not only terrestrial systems but also aquatic systems, such as marine organisms, as a result of adaptive responses that allow organisms to best cope with different environments.Algae, sponges, barnacles, gastropods, bryozoans, and anthozoans are among the marine organisms that have been well studied for their morphological plasticity [9][10][11][12][13].For example, the soft coral Isis hippuris displays distinct morphologies at two different geographical locations within the Wakatobi Marine National Park (WMNP) in Indonesia.In healthy reefs, they present as short-branched with predominantly planar or multiplanar colonies, while being long-branched with bushy colonies in degraded reefs [14].
In addition to widely used molecular methods (e.g., DNA barcoding), chemotaxonomic methods provide an attractive alternative or complementary approach to classifying organisms.This is especially true when dealing with specimens from which purification of intact DNA is difficult or when dealing with symbiotic systems such as sponges and their symbiotic microorganisms [15], which often obscures genetic data.In the following sections, we dissect the utility of chemotaxonomic methods for the classification of the marine red alga Laurencia snackeyi.

Materials and Methods
The literature was retrieved from multiple online databases, such as Google Scholar, and Knowledge Map.Records were searched with keywords related to Laurencia snackeyi and its secondary metabolites.The synonyms employed are Laurencia snackeyi, Laurencia obtusa, Laurencia paniculata var.snackeyi, and Laurencia luzonensis, with a specific emphasis on their secondary metabolites.Any literature that did not report on secondary metabolites was excluded.About 20 records from the years 1980 to 2023 were selected, and their data were extracted and reviewed to assess the feasibility of using biomarkers for taxonomic classification.

Laurencia snackeyi and Its Sesquiterpenes
Laurencia snackeyi (Weber-van Bosse) M. Masuda (1997), which was previously identified as L. paniculata var.snackeyi Weber-van Bosse (1923) and L. obtusa var.snackeyi (Weber-van Bosse) Yamada (1931), is a red alga that can be found in tropical and subtropical waters of the Pacific [16,17].Aside from these scientific names, some of the specimens were tentatively named L. luzonensis before the official adoption of the current nomenclature [18].In this paper, we use the name Laurencia snackeyi (Weber-van Bosse) M. Masuda (1997) to refer to all the abovementioned synonyms.L. snackeyi has been reported to occur in Japan, Sri Lanka, Indonesia, Malaysia, Vietnam, the Philippines, and Australia [16,[18][19][20][21][22].Morphologically, L. snackeyi shares a similar appearance as other Laurencia species but often changes due to environmental factors [23,24].The extensive morphological plasticity of these plants, which hinders conventional classification, has led to extensive studies on the use of sesquiterpenes as potential chemotaxonomic markers.

Chemotaxonomic Value of Halogenated Snyderane-Type Sesquiterpenes in Laurencia snackeyi
The taxonomy of red algae is generally regarded as complex due to their indistinguishable morphological characteristics, which are also often changeable in response to abiotic factors.Chemotaxonomy is thus thought to be a viable alternative solution because these organisms are well known for their unique composition of secondary metabolites.
Generally, a chemotaxonomic marker should possess high specificity and can be consistently identified in specimens collected throughout a species' temporal and spatial distribution.On the basis of the specificity of halogenated snyderane-type sesquiterpenes as proposed markers, the authors found that six out of the ten halogenated snyderane-type sesquiterpenes discovered in L. snackeyi are not specific at the species level, with some of them also reported in other genera.For example, palisadin A (1), which is a halogenated snyderane-type sesquiterpene, was found in other Laurencia species, such as L. implicata J. Agardh (1852), L. similis K. W. Nam & Y. Saito (1991), L. saitoi Perestenko (1980), and L. karlae Zhang et Xia [30][31][32][33].Furthermore, 1 was also reported in non-Laurencia red algae, such as Palisada robusta K. W. Nam (2007) and Ohelopapa flexilis (Setchell) F. Rousseau, Martin-Lescanne, Payri and L. Le Gall (2017) [34,35].Similar phenomena were observed for 2, 4, 5, 7, and 8 (Table 1).While there is a lack of published literature on the remaining four snyderanes (3, 6, 9, and 10), it is highly likely that they can also be found in other red algal species, just like the closely related compounds 1, 2, 4, 5, 7, and 8.According to Cikoš et al. [36], Laurencia sesquiterpenes are interrelated with each other and might arise from the common precursor farnesyl pyrophosphate.The presence of snyderanes in other organisms also indicates the possibility that these red algae relatives possess similar biosynthetic pathways as those found in L. snackeyi.

Chemotaxonomic Value of Halogenated Snyderane-Type Sesquiterpenes in Laurencia snackeyi
The taxonomy of red algae is generally regarded as complex due to their indistinguishable morphological characteristics, which are also often changeable in response to abiotic factors.Chemotaxonomy is thus thought to be a viable alternative solution because these organisms are well known for their unique composition of secondary metabolites.
Generally, a chemotaxonomic marker should possess high specificity and can be consistently identified in specimens collected throughout a species' temporal and spatial distribution.On the basis of the specificity of halogenated snyderane-type sesquiterpenes as proposed markers, the authors found that six out of the ten halogenated snyderane-type sesquiterpenes discovered in L. snackeyi are not specific at the species level, with some of them also reported in other genera.For example, palisadin A (1), which is a halogenated snyderane-type sesquiterpene, was found in other Laurencia species, such as L. implicata J. Agardh (1852), L. similis K. W. Nam & Y. Saito (1991), L. saitoi Perestenko (1980), and L. karlae Zhang et Xia [30][31][32][33].Furthermore, 1 was also reported in non-Laurencia red algae, such as Palisada robusta K. W. Nam (2007) and Ohelopapa flexilis (Setchell) F. Rousseau, Martin-Lescanne, Payri and L. Le Gall (2017) [34,35].Similar phenomena were observed for 2, 4, 5, 7, and 8 (Table 1).While there is a lack of published literature on the remaining four snyderanes (3, 6, 9, and 10), it is highly likely that they can also be found in other red algal species, just like the closely related compounds 1, 2, 4, 5, 7, and 8.According to Cikoš et al. [36], Laurencia sesquiterpenes are interrelated with each other and might arise from the common precursor farnesyl pyrophosphate.The presence of snyderanes in other organisms also indicates the possibility that these red algae relatives possess similar biosynthetic pathways as those found in L. snackeyi.
On the basis of the consistency of halogenated snyderane-type sesquiterpenes across study periods and locations, some studies have isolated as many as five types of snyderanes and some just two types (Table 2), calling into question the viability of using these metabolites as chemotaxonomic markers.Moreover, inconsistencies can be observed in the ratio of the yielded snyderanes.While these inconsistencies may result from various abiotic and technical factors, an important trait of an ideal marker is that it should be easily measured and has an almost foolproof protocol.The detection and identification of sesquiterpenes are carried out mainly via spectroscopic techniques such as nuclear magnetic resonance, which results in lower resolution and often requires large amounts of purified compounds for analysis [39], further hindering the use of these metabolites as potential chemotaxonomic markers.Furthermore, the use of mass spectrometry for detecting these metabolites may yield "unidentified" results due to the constraints in matching databases for identification.
As evidenced above, the utility of sesquiterpenes as potential chemotaxonomic markers for L. snackeyi or among Laurencia spp. is questionable because these compounds lack specificity and consistency.Interestingly, but not unexpectedly, a similar phenomenon has been observed in other studies on algae and plants that claim to have discovered certain classes of metabolites as chemotaxonomic markers [36,40,41].In addition to snyderanes, there are other classes of sesquiterpenoids that have been claimed to be species-specific.According to Palaniveloo and Vairappan [22], L. snackeyi produces snyderanes, L. majuscula produces chamigranes, L. similis produces bromoindoles, and L. nangii produces acetogenins.However, based on our literature search, chamigranes [28], cuparanes [18,29], and monocyclofarnesol-type sesquiterpenoids [22,27,29,42] have also been found in L. snackeyi, which raises concerns about the reliability of other chemotaxonomic markers for other Laurencia species.

Conclusions
In the case of L. snackeyi, chemotaxonomy based on secondary metabolites lacks reliability and consistency because the proposed halogenated snyderane sesquiterpenes are not species-specific, as claimed in previous studies.Along with the advancement of highthroughput metabolite profiling tools and data-processing platforms, chemometric-based chemotaxonomy could be a game changer.Nuclear magnetic resonance and mass spectrometry metabolomics, which combine high-throughput analytical tools with multivariate data analysis, are gaining popularity in chemotaxonomic analysis [44][45][46][47].High-throughput metabolomic-based chemotaxonomic analysis will capture and interpret metabolome fingerprints without the need for metabolite identification or structural analysis.Chemometric data, such as metabolomics approaches, would be sufficient for species identification and characterization [48][49][50].Similarly, the processed chemometric data could be matched to a database for taxonomic classification and qualitative studies of the potential bioactivities of the extracts.

Figure 1 .
Figure 1.The halogenated snyderane-type sesquiterpenes that have been identified from the L. snackeyi.

Figure 1 .
Figure 1.The halogenated snyderane-type sesquiterpenes that have been identified from the L. snackeyi.

Table 2 .
Reported yields of the halogenated snyderanes isolated from L. snackeyi since 1997.