Sponge Spicules in Sediments: A Proxy for Past and Present Sponge Fauna Assessment in Ha Long Bay (Vietnam)

Abstract

The study of cryptic sponges can be challenging, as they are difficult to detect in the environment without the use of destructive methods. The permanence of mineral skeletal components of Porifera in sediments is therefore a tool that can provide support for a more comprehensive study of sponge fauna. In this context, sediments collected from four karst lakes in Ha Long Bay (Vietnam), plus two surrounding sea areas, were processed to extract spicules belonging to sponges with siliceous skeletons. From the morphology of the spicules, it was possible to trace the taxa of the sponges present in the area at that time or earlier. It was found that each lake had a sponge fauna composition characterized by different families and genera. Moreover, sponge taxa identified in the surrounding sea areas differed significantly from the ones inside of the neighboring lakes. In addition, it was possible to identify the presence of four sponge genera not yet recorded in the South China Sea area. Thus, the study of spicules trapped in sediments proved to be a useful tool for a comprehensive study of sponge fauna.

1. Introduction

Sponges have received much attention in recent years due to the recognition of their multiple ecological roles, diversity, and abundance [1,2,3]. Nevertheless, the diversity of sponge fauna is often underestimated because, alongside conspicuous and large species, many others are small or cryptic, living inside the crevices of substrate or boring inside it. This “hidden diversity”, which is difficult to estimate, may constitute the larger part of the total diversity [4].

While massive and large encrusting species can often be easily identified in the environment, boring and cryptic sponges may be difficult or even impossible to detect and need to be collected by removing part of the substrate [5,6,7]. Since sampling of hard substrate is a time-consuming and necessarily destructive method, it is therefore ideal to avoid this technique, if possible, especially in sensitive and protected environments. For these reasons, the study of Porifera spicules, which are incorporated in sediment after the sponge’s death, is a valid alternative possibility for the detection of cryptic species [8] or species recently prone to mortality or reduced in biomass. In fact, while the organic component of sponges undergoes physical and biological processes that lead to decomposition, spicules remain intact for a longer time due to their mineral composition [9,10], depending on the mineral skeleton composition, calcareous or siliceous, and the environmental conditions [9,11]. Sponge spicules are identified by their morphology, and while some of these are shared by several taxa, others are characteristic of a specific genus and play an important diagnostic role [8,12]. Therefore, joining the study of living species of Porifera, together with the analysis of spicules in the surrounding sediment, can be a more comprehensive method to investigate the sponge fauna diversity of a selected habitat [13].

Ha Long Bay (Vietnam) is located in the northern area of the Gulf of Tonkin in the South China Sea. This environment is characterized by the presence of numerous karst islands where tropical weather conditions facilitated the formation of shallow salt lakes [14] hosting unique community assemblages, including the porifera fauna present [15,16]. These karst marine embayments, commonly defined as lakes, are often poorly connected with the outer marine area, making them a semi-enclosed environment [17]. Moreover, they are characterized by seasonal environmental conditions which induce sponge growth in winter and spring followed by a degeneration during summer, when the temperature can exceed 38 °C [18]. The lakes of Ha Long Bay are therefore an ideal place to study sponge spicules in the sediment, which are expected to remain largely confined within the basins. Furthermore, the sponge fauna of Vietnam has not been intensively investigated; thus, limited knowledge of this benthic component exists for the area [15,16,18,19,20,21,22,23,24,25].

The present paper aims to reveal the possible hidden sponge diversity of Vietnamese lakes using the spicules accumulated in their sediments.

2. Materials and Methods

Sampling took place in August 2018 in 4 different lakes of Ha Long Bay (Vietnam), namely Cahong, Hang Du I, Bui Xam, and Me Cung (Figure 1). The marine lakes selected for this study are characterized by a different degree of connection with the surrounding sea. In fact, some of them are closer to the coast and affected by tidal currents, while others are far from the coast and completely isolated except for the presence of karst porosity. Indeed, the study sites can be categorized as semi-enclosed lakes (Me Cung) and enclosed lakes (Cahong, Hang Du I, and Bui Xam) [16,17]. The salinity of the water in the surrounding sea is around 32‰, while temperature ranges from a minimum of 23 °C in winter to a maximum of 30 °C in summer, and tidal range is 3–4 m [16]. In marine lakes, water temperature can be even higher, reaching peaks of 38 °C [18]. The weather conditions in the area are typically tropical and characterized by an annual average air temperature of 25 °C and monsoons which are hot and humid in summer and cold and dry in winter [15].

Sediments were sampled by SCUBA diving, taking a core measuring 10 cm in length and 3 cm in diameter. Sampling was carried out at two locations for each lake, at a depth of 1 m and 4 m, respectively. Furthermore, sampling of sediment was also carried out in the seabed in the external areas surrounding Hang Du I and Bui Xam lakes, at depths of 1 m and 4 m. Three replicates were collected for each depth and site. In the laboratory, the sediments were dried and weighted. For each replicate, a subsample of 0.5 g of sediment was heat-treated with 65% nitric acid to isolate siliceous spicules of Porifera from the carbonatic fraction. The treatment was carried out by renewing the nitric acid at least once during the process, which lasted as long as it was required to dissolve all the carbonate components. This method made it possible to detect only the spicules of any Hexactinellida or Demospongiae and Homoscleromorpha with opaline silica skeletons. The material, resulting from nitric acid treatment, was analyzed by light microscopy to identify sponge spicules and consequently the taxa to which they belong [26]. Only the complete spicules were considered, and all complete spicules were counted for each subsample. For many spicules, it was possible to determine taxonomy to the genus level due to unique diagnostic features, while in other cases it was possible to assign spicules to the family or order level. Comparisons were carried out between sponge genera identified by analysis of spicules and those already known from the same lakes thanks to previous extensive benthic sampling [16].

Two-way ANOVA was performed to test the differences between different areas and depths, in terms of number of families/genera of sponges identified from the spicules found, followed by Tukey test for pairwise comparison. The same analysis was carried out to compare density of spicules in sediment samples. To investigate dissimilarities in the presence of the various families/genera in different areas, a NMDS was applied, followed by the ANOSIM test and Indicator Species Analysis (ISA). Statistical analysis was carried out by R studio software (2022.07.2 Build 576).

3. Results

3.1. Comparisons Between Sampling Areas

The average concentration of spicules in sediment from the lakes was as follows: 3028 ± 4045 spicules/g in Cahong, 1178 ± 308 spicules/g in Hang Du I, 1604 ± 124 spicules/g in Bui Xam, and 2106 ± 195 spicules/g in Me Cung. In the marine areas surrounding Hang Du I and Bui Xam lakes, the mean values of spicule concentrations were 1538 ± 427 and 1838 ± 710 spicules/g, respectively. Overall, most of the spicules belonged to sponges of the class Demospongiae (Heteroscleromorpha), among which a total of 11 orders, 24 families, and 28 genera were identified (

Table 1. List of taxa found in analyzed spicules and their presence (+) in the study areas.

Class	Subclass	Order	Family	Genus	Cahong	Hang Du I	Bui Xam	Me Cung	Hang Du I Out	Bui Xam Out
Demospongiae	Heteroscleromorpha	Agelasida	Agelasidae	Agelas				+
		Axinellida	Raspailiidae	Echinodictyum					+
		Biemnida	Biemnidae	Biemna		+	+	+	+	+
			Rhabderemiidae	Rhabderemia		+	+	+	+	+
		Bubarida	Desmanthidae	Desmanthus			+		+	+
		Clionaida	Clionaidae	n.a.	+	+	+	+	+	+
				Cliona	+	+	+	+	+	+
				Cliothosa		+	+	+	+
				Dotona			+	+	+	+
				Pione				+	+	+
				Spheciospongia		+	+
				Spiroxya		+	+		+	+
			Placospongiidae	Placospongia	+	+	+	+	+	+
			Spirastrellidae	Spirastrella	+		+		+
		Haplosclerida	n.a.	n.a.		+		+	+
			Chalinidae	n.a.		+	+	+	+	+
			Petrosiidae	Petrosia		+	+	+	+	+
				Xestospongia		+	+	+	+	+
			Phloeodictyidae	Siphonodictyon			+	+	+	+
		Poecilosclerida	n.a.	n.a.	+	+	+	+	+	+
			Acarnidae	Acarnus			+	+	+	+
				Zyzzya					+	+
			Coelosphaeridae	Coelosphaera				+		+
			Crellidae	n.a.			+
			Mycalidae	Mycale		+	+	+	+	+
			Myxillidae	n.a.		+	+	+	+	+
			Tedaniidae	Tedania	+	+	+	+
		Spongillida	Spongillidae	Spongilla	+
		Suberitida	Halichondriidae	n.a.		+	+	+	+	+
			Suberitidae	n.a.	+	+	+	+	+	+
				Aaptos			+	+
				Protosuberites	+	+	+
				Terpios	+	+	+	+	+	+
		Tethyida	Tethyidae	Thethya		+	+		+	+
		Tetractinellida	n.a.	n.a.	+	+	+	+	+	+
			Ancorinidae	Stelletta			+	+		+
			Geodiidae	n.a.	+	+	+	+	+	+
			Samidae	Samus			+	+	+	+
	Verongimorpha	Chondrillida	Chondrillidae	Chondrilla		+			+
Homoscleromorpha		Homosclerophorida	Plakinidae	Plakina			+	+	+

). Only one representative of the subclass Verongimorpha, the genus Chondrilla, was found out. The genus Plakina was instead the only one found belonging to the class Homoscleromorpha (Table 1).

The abundance of spicules in sediment samples was not significantly different between lakes (p > 0.05), nor was it when comparing samples from the inside and outside of Hang Du I and Bui Xam lakes. Worth mentioning is the fact that in samples from Cahong, most of the spicules belonged to the genus Spongilla.

Differences in terms of the number of families/genera were found to be statistically significant between lakes (two-way ANOVA p < 0.001) but not between different depths within each lake (p > 0.05) (Figure 2). Furthermore, no significant difference was found in the abundance of families/genera between the inside and the outside of Hang Du I and Bui Xam lakes (p > 0.05). Spicules found in sediments indicated the highest diversity of sponge taxa in Bui Xam lake and the lowest in Cahong lake.

The spicules found in lake sediments were characterized by the presence of a different pool of sponge families and genera depending on the area, as highlighted by the NMDS and confirmed by the ANOSIM test (R: 0.6, p < 0.001 between areas) (Figure 3). In addition, in the areas of Hang Du I and Bui Xam lakes, the type of families/genera varied between the inside and outside (ANOSIM test—R: 0.4, p < 0.001) (Figure 4).

3.2. New Findings for the Area of the South China Sea

A comparison was carried out between sponge genera identified by analysis of spicules, with those observed in living sponges from the same sampling campaign [16], and those from published check lists of the area [24,27,28]. Among the 30 genera identified by spicules in sediment, 10 were already known for the Ha Long Bay area [16], 16 were not found in the studied lakes but are recorded in check lists of the Vietnam coasts and Tonkin Gulf [24,27,28], and 4 are new findings for the area of the South China Sea (Figure 5) (Table S1). These last are as follows: Dotona Carter, 1880, Zyzzya Laubenfels, 1936, Samus Gray, 1867, and Plakina Schulze, 1880 (Figure 6). The spicule morphotypes belonging to genera as yet unnoted from the South China Sea are described below.

3.2.1. Spiral Spined Strongyles of Dotona Carter, 1880

A total of 50 spiral-spined strongyles were found in Me Cung and Bui Xam lakes and in the marine areas outside Hand Du I and Bui Xam lakes. Most of those spicules were in sediment samples collected outside Hang Du I lake. Spines were distributed all along the rhabd following a spiral pattern, and the spicules had rounded ends (Figure 6A).

Remarks: The genus Dotona is poorly represented, with only two species distributed in the Indian Ocean and Pacific Ocean and a Mediterranean subspecies [29]. The habitus is boring [30]. Micro-spined strongyles are characteristic microscleres of the genus, which may be bent or undulated and most commonly patterned in a spiral around the rhabd [30]. Despite spiral spined strongyles being established to also be present in the genus Spiroxya [31], they are only recorded in the species Spiroxya heteroclita Topsent, 1896, which is strictly Mediterranean [29]. Indeed, the only two species of Spiroxya recorded in the study area are Spiroxya acus (Bavestrello, Calcinai, & Sarà, 1995) and Spiroxya macroxeata (Calcinai, Bavestrello, Cerrano, & Sarà, 2001) [28], which do not show the presence of spiral spined strongyles [32,33].

3.2.2. Verticillately Spined Strongyles of Zyzzya Laubenfels, 1936

Verticillately spined strongyles were detected in sediment collected in the marine areas outside Hang Du I and Bui Xam lakes: only one spicule of this type was found in each site. The spicules had a markedly pointed termination. The spines were distributed all along the shaft and vertically arranged to form 18–20 whorls (Figure 6B).

Remarks: The genus Zyzzya is characterized by the presence of accessory spicules consisting of verticillately spined strongyles (or strongyloxeas), which form an irregular reticulation interspersed with the main choanosomal skeleton [34]. The habitus range from fistulose massive to cryptic encrusting [34] and boring [5]. Five species represent this genus, mostly Indo-Pacific [29].

3.2.3. Amphiplagiotriaenes of Samus Gray, 1867

The amphitriaenes characteristic of the genus Samus were found in Me Cung and Bui Xam lakes and in the marine areas outside Hand Du I and Bui Xam lakes, for a total of seven spicules. The spicules found were short-shafted amphiplagiotriaenes, with clads distally recurved and forming an angle of about 45° with the rhabd. Cladome were stout with short deuterocladi, often dichotomously divided (Figure 6C).

Remarks: The genus Samus is the only representative of the family Samidae, and only one cryptic species complex, namely Samus anonymus Gray, 1867, is recognized as belonging to it [35]. Specimens have limited size and a boring habitus and possess a skeleton which consist of small amphitriaenes and sigmaspires [35]. Both amphipro- and amphiplagiotriaenes may occur, and symmetrical or asymmetrical geometry depends on dichotomous or trichotomous division of cladi [8]. The distribution is wide and includes the Mediterranean Sea, Caribbean, and Atlantic Ocean [29].

3.2.4. Homolophose Calthrops of Plakina Schulze, 1880

A total of six homolophose calthrops, indicating the presence of the genus Plakina, were detected in sediment from Bui Xam and Me Cung lakes and from the marine areas outside Hand Du I lake. Spicules were monolophose calthrops with one actine homogeneously ramified in three to four secondary rays (Figure 6D,E).

Remarks: The genus Plakina is characterized by the presence of homolophose calthrops with one, two, three, or four lophate rays, complementing the main skeleton of non-lophose spicules, such as diods, triods, and calthrops [36]. The habitus is thinly to massively encrusting [36]. This genus is widely distributed, comprehending both warmer waters, such as the Mediterranean Sea and the Caribbean, and colder oceanic areas, from the North Atlantic to the South Atlantic and Pacific to the ice-cold waters of Antarctica [29].

4. Discussion

The four lakes studied in Ha Long Bay were significantly different in the number and diversity of sponge taxa. The highest number of both family and genera was found in Bui Xam lake, followed by Me Cung lake, Hang Du I lake, and Cahong lake. The same ranking occurred for the analysis of living sponges [15], and it does not seem to be related with the degree of connection with the open sea, Me Cung being the only semi-enclosed lake, while the others were completely enclosed [16,17]. Instead, physical characteristics, such as water column stratification and sedimentation rates, are likely to have the greatest influence on sponge fauna [37,38]. In fact, Cahong lake, which showed the lowest diversity of sponge taxa, is characterized by wide seasonal temperature fluctuation, with particularly high values reached in summer at the lake bottom and low salinity [18]. Indeed, previous studies indicated that Cahong lake hosted only one species of Demospongiae, which was the freshwater sponge Spongilla manconiae Calcinai & Bertolino, 2020 [16,18]. In accordance with those findings, the Indicator Species Analysis (ISA) highlighted the prevalence of spicules from the Spongillidae family in sediments from Cahong lake, but this was not the only taxa found. In this area, spicules also showed the presence of the families Clionaidae, Spirastrellidae, Suberitidae, Tedaniidae, and Geodiidae, together with spicules from the orders Astrophorina and Poecilosclerida. Therefore, the data from the sediment analysis of Cahong lake were particularly interesting, showing the presence of sponges which were not previously detected. However, in Cahong almost all the spicules found belonged to the genus Spongilla, due to the massive presence of the only living species detected, S. manconiae [18]. The fact that only specimens of the new Spongilla species were found in the sampling of living specimens could be because that survey was carried out in the warm season. This is one of the factors that could explain the failure to find other living sponge species, which were instead identified through the analysis of spicules in the sediments. Indeed, it has been reported that some sponge species of the lakes disappear during the harsh hot conditions and reappear at the end of the warmer season [17]. This mechanism has also been described in the Adriatic Sea for the species Tedania (Tedania) anhelans (Vio in Olivi, 1792) that almost disappear in autumn and winter [39]; similarly, Corticium candelabrum Schmidt, 1862 shows a seasonal growth cycle, undergoing mortality events during cold months in the northwestern Mediterranean Sea [40].

Regarding Hang Du I lake, the scarce diversity of sponge taxa was already highlighted by previous studies, which blamed severe water stratification occurring in the area [15,16]. As before, Hang Du I was mainly characterized by sponges of the family Suberitidae and a high presence of Tethyidae. Nonetheless, the analysis of sediment spicules showed the sporadic occurrence of sponge genera which were not previously detected in the lake, such as Biemna, Mycale, Xestospongia, and Placospongia. Bui Xam lake had the highest diversity of sponge taxa in the sediment spicules and was even higher than Me Cung lake, although the latter seems to be more connected to the open sea [17]. It is possible, however, that there is a greater connection to the open sea at Bui Xam that has not yet been identified but can be assumed due to the presence of massive corals [15]. However, the number of taxa identified by spicule analysis was not markedly different from that of studies on live sponges [15,16], but it does provide additional information. For example, among the genera that, according to the ISA, mostly characterized the sponge fauna of Bui Xam were Rhabderemia and Placospongia, which were not previously detected in the area [15,16]. Furthermore, the sediments both inside and outside this lake showed the presence of spicules from the genus Dotona, which is a new finding for the South China Sea. As for Me Cung, the ISA showed the highest characterization due to spicules of the families Chalinidae and Halichondriidae, but no genus belonging to the latter has been previously found within the lake [15,16].

The spicules in the sediments of Hang Du I and Bui Xam lakes showed diversity, in terms of the number of sponge taxa, similar to that of coastal areas outside each lake. However, the NMDS and ANOSIM tests revealed a qualitative difference between the inside and the outside of each site. Therefore, the scarce exchange of water with the open sea did not prevent the formation of diverse sponge fauna within the enclosed lakes, but it did affect its specific composition. In fact, marine lakes are known to be characterized by a peculiar and sometimes unique fauna, depending on their degree of connection with the open sea, not only in the Vietnam area but also in Indonesian regions [41].

A total of 30 genera of sponges were found among the spicules of sediments, more than half of which were not found among the living sponges reported for the studied lakes [15,16,18]. Most of these are known to be present in the South China Sea, but four genera, which are Dotona, Zyzzya, Samus, and Plakina, are new findings for the region [24,27,28]. Their absence in literature records could be due to their mostly cryptic habitus.

The presence of spicules belonging to sponge taxa that are not detectable in the environment as living organisms is therefore important information but should be cautiously considered. In fact, free sponge spicules in soft superficial sediments already show signs of dissolution after 6 months, and they completely dissolve in two years [9]. This process depends on physical factors, such as pH and temperature of water. For example, it is well known that spicules can be preserved for decades within the mats of Posidonia oceanica (Linnaeus) Delile, 1813, and even millennia in sediments trapped inside coralligenous concretions [9]. Moreover, the organisms responsible for bioturbation can cause the sediment to be mixed in a layer up to 20–40 cm thick, resulting in a mixture of material that may have been deposited at different times, depending on the rate of sedimentation [8]. Therefore, spicules in the sediment may indicate the presence of an organism currently in the environment or the former presence of the sponge. The difficulty in distinguishing the age of sediment layers on soft bottoms is in fact due to both physical and biological factors. Moreover, as these areas are characterized by periodic monsoon events, and as the lakes are located close to the outer sea, it is still possible for material to be transported from the outside to the inside of the lakes. These are therefore all factors that must be taken into account when carrying out this type of study. Currently, another method for detecting the presence of cryptic species is the analysis of environmental DNA (eDNA), which, however, presents limitations related to its faster degradation, which occurs due to both chemical and biological processes [42]. In conclusion, the study of spicules in sediments can be a very useful tool for a more in-depth study of past and present sponge fauna, especially in closed or semi-closed environments such as the lakes of Ha Long Bay.