Abstract
Background Red calcareous seaweeds of the genus Galaxaura have been associated with coral reef degradation. Galaxaura has low nutritional value for most herbivorous fishes and produces allelopathic chemicals in competition with corals. In this study, the abundance of the filamentous Galaxaura divaricata was surveyed on 13 spatially independent patch reefs across the lagoon of Dongsha Atoll, an isolated coral reef ecosystem in the northern South China Sea. Variations in Galaxaura cover on a degraded reef were monitored over a period of 17 months. Epiphytic macroalgae associated with Galaxaura were quantified, and species identifications aided through DNA barcoding.
Results Patch reefs in the lagoon of Dongsha Atoll were degraded, exhibiting relatively low living coral cover (21 + 3%), but high cover of macroalgae (30 + 4%), and rubbles and dead coral (47 + 4%). The distribution of Galaxaura was heterogeneous across the lagoon, with highest abundance in the southeast area. In that area Galaxaura has persistently bloomed on degraded reef for at least four years, covering up to 41% of the substrate. Galaxaura provides substrate for various macroalgae, 15 of which were identified to the species level, four to the genus level, one to the order Gelidiales, and one to the phylum cyanobacteria. Some of these epiphytes are allelopathic and known to frequently overgrow corals. For instance, the brown alga Lobophora sp28, third most common epiphytic macroalga on Galaxaura, frequently overgrows corals across the lagoon of Dongsha Atoll.
Conclusions Our study demonstrated that an allelopathic and unpalatable seaweed, such as Galaxaura can bloom on degraded coral reefs for several years. The complex thallus-structure of Galaxaura provides suitable substrate for other macroalgae, some of which are noxious to corals. By increasing the substrate availability for macroalgae, Galaxaura could facilitate the abundance of macroalgae, and decrease the recovery potential of degraded coral reefs.
Introduction
Coral-macroalgae competition is a major ecological process on coral reefs [1,2]. However, climate change, overfishing, and anthropogenic pollution have driven corals worldwide to a decline and often facilitated macroalgae dominance on degraded reefs [3-9]. The replenishment of corals on degraded reefs is strongly influenced by the types of dominant macroalgae [10,11]. Macroalgae that produce noxious allelochemicals in competition with corals are considered most detrimental for the resilience of degraded coral reefs [10,12-14]. Allelopathic macroalgae may perpetuate their dominance on degraded reefs by inhibiting the process of successful recruitment of juvenile corals, the key process of coral reef recovery [15-17].
Species of the red, calcareous genus Galaxaura are common on coral reefs in the Pacific Ocean [18-22]. Galaxaura is one of the least nutritious macroalga to herbivores fish [23-26], and highly competitive with coral because of its allelopathic effects on coral and coral larvae [15,27]. Extracts of the lipid-soluble secondary metabolites of Galaxaura filamentosa were shown to cause bleaching and death of coral tissue upon direct contact [13-15,28], and deterred coral larvae from settlement [29]. It has been suggested that high abundance of Galaxaura on degraded reefs accounts for the inhibition of coral reef recovery [12,29]. More detailed investigations of the distribution and ecology of reef-associated seaweeds, such as Galxaura, and their roles in outcompeting corals will provide valuable information for coral reef management and conservation [1,30].
Dongsha Atoll (also known as Pratas Islands) is the only large coral reef atoll (>500 km2) in the northern South China Sea. The South China Sea is the largest tropical Pacific marginal sea in Southeast Asia [31], including numerous coral reef atolls and fringing reefs that are great natural resources, supplying large numbers of people with goods and services, such as fish and important ecological values [32-34]. The ring-shaped reef flat of Dongsha Atoll encircles a large lagoon with seagrass beds and hundreds of coral patch reefs [35]. The Lagoon patch reefs are structured into tops (1-5 m depth) and slopes (5-12 m depth), and provide important habitat and sheltered nursery grounds for numerous marine organisms, such as green sea turtles and coral reef fish, including rays and sharks [32,35-37]. The catastrophic mass bleaching event in 1998 and reoccurring bleaching events thereafter have caused severe mass mortalities of corals in the lagoon, followed by a marked increase of macroalgae [36,38].
The filamentous, calcareous macroalga Galaxaura divaricata is conspicuous on coral reefs in the lagoon of Dongsha Atoll [19] where it blooms in certain areas, overgrowing large parts of degraded reef substrate. Several macroalgae, e.g. Sargassum and Lobophora, are known to provide substrate and form habitats for epiphytic algae [39-41]. Increased substrate availability can lead to increased macroalgae biomass on coral reefs [1]. However, whether Galaxaura provides a suitable substrate for macroscopic epiphytes remains unexplored.
The aims of this study are to document the spatial patterns and persistence of Galaxaura blooms in the lagoon of Dongsha Atoll, show several aspects of Galaxaura-coral competition, and identify and quantify epiphytic macroalgae that grow on Galaxaura. Potential implications of our observations, regarding the recovery potential of Galaxaura dominated reefs, and the role of Galaxaura as substrate for the facilitation of macroalgae abundance, are discussed.
Materials and methods
This study was conducted from April 2016 to September 2017 in Dongsha Atoll (Pratas Island) (Latitude 20°40’43”N, Longitude 116°42’54”E), an isolated coral reef atoll in the north of the South China Sea. The climate shows seasonal variability under the East Asian monsoon system, with a colder northeast monsoon season (as winter) and a warm southwest monsoon season (as summer) [31]. The ring-shaped atoll covers an area of approximately 500 km2 and is situated 450 km southwest from the coast of Taiwan and 350 km southeast from Hong Kong (Fig 1). The outer reef flat is interrupted by two channels that are located north and south of a small islet (1.74km2), allowing for water exchange between the lagoon and the open ocean [36,42]. The semi-closed lagoon is about 20 km wide with a maximum depth of 16 m deep near the center [36].
The abundance of the red, calcareous, filamentous seaweed Galaxaura divaricata was surveyed on 13 spatially independent lagoon patch reefs with SUBA (Fig 1B and S1 Table). Two 45-m transect lines were laid out across the reef top (1-5 m depth) and the reef slope (5-12 m depth) respectively. The percent cover of Galaxaura, living coral, macroalgae (MA; including low growing, filamentous turf algae [23, 24]), crustose coralline algae (CCA), and other substrate was estimated, using a 35 cm × 50 cm PVC quadrat [43]. Estimations were done at 1 m intervals and a total of 90 replicate quadrats were analyzed for each site. ‘Other substrate’ mainly constituted dead corals, rubbles, larger rocks covered with sediments and sparse turf algae, and sand. The seawater temperature was measured every 30 min from March 2016 to September 2017, using Hobo temperature loggers that were fixed at the reef top and reef slope of each site.
To assess variations over time we monitored the percent cover of Galaxaura and living coral across the slope of site 7 at 5 m depth in April, July, and September 2016 and in September 2017. 45 replicate photos were taken with an Olympus Stylus-TOUGH TG4 digital camera (25-100 lens, 35mm equivalent) mounted onto a PVC-quadrat at 0.64 cm above a sampling 35 cm × 50 cm quadrat [43], using the survey method described above. Percent cover estimations of Galaxaura were aided by superimposing a 10 × 10 reference grid onto each photograph, where 1 quadrat represented 1% of the total area.
To quantify epiphytic macroalgae associated with Galaxaura, we randomly collected 30 thalli of Galaxaura from site 7 at 5 m depth. The samples contained an equal proportion of small (6 + 4 g), medium (23 + 6 g), and large (49 + 20 g) thalli. Epiphytic macroalgae were identified to the closest identifiable taxonomic unit, using either the guidebook [19] or DNA barcoding. The presence and absence of each taxonomic unit was recorded, and the occurrence frequency (f) was calculated as follows: f = c (taxonomic uniti)/n, where c (taxonomic uniti) stands for the count number of thalli that have the epiphyte taxonomic unit i, and n stands for the total number of thalli analyzed.
Two-paired t-tests were used to detect significant differences of live coral, macroalgae, and CCA percent cover between reef tops and reef slopes. A two-way ANOVA and a post-hoc Tukey test were applied to test for significant differences of Galaxaura percent cover between reef top and reef slope and among sites. Percent cover of Galaxaura and living coral cover at site 7 from four surveys conducted over a 17-month period were analyzed using a two-way ANOVA, and p-values < 0.05 were considered significant.
Macroalgae samples were preserved in silica gel and species identified through DNA barcoding [44]. DNA was extracted with Quick-DNATM Plant/Seed Miniprep Kit (Zymo Research Co., USA). Primers for the plastid gene specific amplifications were used as follows: rbcL F7/R753 for rhodophytes [45], rbcL F68/R708 for Phaeophyceae [46], and tufA F210/R1062 for green algae [47]. The newly generated sequences were deposited in GenBank and searched using BLASTn against the GenBank database (S2 and S3 Tables). Sequence similarities of > 98% were considered for species identification.
Results
Seawater temperatures in the lagoon were highest during the southwest monsoon season (June-Sept.), averaging 30.1°C, and lowest during the northeast monsoon season (Nov.-April), averaging 24.8°C. In July and August, maximum temperatures reached 34°C on reef tops and 32.7°C on reef slopes. Across sites the average coral cover accounted for 21 + 3% (+ SE) of the benthic substrate (range: 5-43%) (Table 1). Average coverage of the substrate was 30 + 4% (range: 13-58%) by macroalgae, and 2 + 0.2% (range: 1-3%) by crustose coralline algae (CCA). Dead corals, rubbles, larger rocks, and sand accounted for an average of 47 + 4% (range: 23-69%) of the substrate. Living corals dominated the reef substratum on two out of 13 patch reefs (sites 1 and 13), whereas dead corals, rubbles, larger rocks, and sand dominated the substrate on nine out of 13 sites. Macroalgae cover exceeded live coral cover on seven out of 13 sites. No significant difference in coral cover and macroalgae cover between reef tops and reef slopes was detected (P-values = 0.394 and 0.552).
The percent cover of Galaxaura divaricata was significantly different between reef tops and reef slopes (P-value < 0.01), as well as among the 13 survey sites (P-value < 0.001). The post-hoc Tukey test showed that Galaxaura was more abundant on reef slopes than on reef tops (P-value <0.05). The percent cover of Galaxaura was classified as very low (0-1.5%), low (1.5-5%), moderate (5-20%), and high (>20%) (P-value < 0.05; post-hoc Tukey). Galaxaura was most abundant on patch reef sites in the southeast lagoon (Table 2). The percent cover of Galaxaura was highest at site 9 (41%) and on the slope of site 7 (16%). Patch reefs in the northeast lagoon exhibited moderate, low, and very low cover of Galaxaura (range: 0.21-5.7%). Survey sites in the south, center, west, and north section of the lagoon were characterized by very low cover of the alga (range: 0-1.4%). The thallus shape and size of Galaxaura varied across sites (S1 Fig). Medium (5-15 cm diameter), ball-shaped thalli and large (15-30 cm), carpet-like thalli were exclusively present in the southeast lagoon. Small (1-5 cm), ball-shaped thalli and small, slender thalli were dominant on patch reefs in the northeast lagoon. DNA barcodes revealed that all samples of Galaxaura from various sites across the lagoon were 100% identical in their rbcL sequences, indicative of conspecificity (S3 Table).
Our observations suggest that a Galaxaura bloom can persist for several years. Thick canopies of Galaxaura have overgrown dead Acropora rubbles for at least 4 years on a degraded patch reef in the southeast section of the lagoon, with 41 + 25% Galaxaura cover and 5 + 14% living coral cover (site 9, 3-5 m depth) (Fig 2A-B). Galaxaura was frequently observed in contact with coral, which in some cases showed beached and dead tissue underneath or in contact with Galaxaura. The holdfast of Galaxaura penetrates the calcium carbonate structure, creating a strong attachment to the coral (Fig 2C). In some cases, fluorescent pink and bleached coral tissue occurred in contact with Galaxaura, indicative of potential allelopathic effects of G. divaricata on corals (Fig 2D).
The percent cover Galaxaura did not vary significantly among surveys at site 7 conducted over a period of 17 months (Fig 3). The cover of Galaxaura and living corals did not change significantly among surveys (P-value = 0.595). Galaxaura cover averaged 15.91 + 0.6% (+ SE), and coral cover remained low, averaging 16.45 + 1.17%. Coral cover and Galaxaura cover were not significantly different (P-value = 0.770), and no interaction between the two was detected (P-value = 0.780).
In our epiphyte survey we identified 21 taxonomic groups of macroalgae in association with Galaxaura. Among those groups, we identified 15 to the species level, four to the genus level, one to the order Gelidiales and one to the phylum cyanobacteria (Table 3 and S2 Table). Among the 15 identified species, red algae were most abundant (10), followed by brown algae (5), and green algae (5). The most common species associated with Galaxaura were the red algae Hypnea caespitosa [48] (100% relative abundance) (Fig 4D), Coelothrix irregularis (87%), Ceramium dawsoniia (43%) and the brown algae Lobophora sp28 [49] (57%) (Fig 4D), Padina sp5 [50] (53%), and Dictyota bartayresiana (30%). The most common green algae associated with Galaxaura were Derbesia marina (37%), Caulerpa chemnitzia (27%) (Fig 4B), and Boodlea composita (20%). Epiphytic macroscopic cyanobacteria (filamentous > 1cm) had a relative abundance of 17%. Lobophora sp28, third most common macroalga on Galaxaura, was identified to frequently overgrow corals across the lagoon of Dongsha Atoll (Fig 5 and S2 Fig).
Discussion
The results of the benthic surveys indicate that coral reefs in the lagoon of Dongsha Atoll are highly degraded, with high abundance of dead corals, rubbles, larger rocks, and sand (47%). Our observation is consistent with previous surveys that noted signs of coral degradation in the lagoon, with an extremely large proportion of coral rubbles and dead corals [36,51,52]. The majority of patch reef sites exhibited a coral cover of lower than 25%, which is considered degraded [53]. Coral reefs in the South China Sea have been facing increasing chronic and acute thermals stress over the past decades [33,38,51,54]. The summer mean sea surface temperature shows an average upward trend of 0.2 / decade, with waters surrounding Dongsha Atoll warming at a faster rate than other areas of the South China Sea [33]. Prior to the 2007 establishment of Dongsha Atoll Marine National Park, Dongsha Atoll was heavily overfished, and large coral reef areas were destroyed through dynamite and cyanide fishing [36,52,54,55]. According to a previous survey coral cover and the number of new small corals were especially low on patch reefs in the south section of the lagoon [56]. Recurrent coral bleaching events [36,38,57], coral damage by annual typhoons, and destructive fishing practices may have acted synergistically, leading to the failure of recovery for some of the lagoon patch reefs. Coral cover and coral recruitment rate was especially low on patch reefs in the southeast lagoon [56].
Species of the red calcareous alga genera Galaxaura occur in two morphological forms, a smooth and a filamentous form, which is characterized by fine, assimilatory filaments [21]. Our observations suggested that, under certain conditions, the filamentous Galaxaura divaricata can become dominant on degraded reefs and form long-standing canopies that grow frequently in contact with corals. Galaxaura was heterogeneously distributed across the lagoon, and bloomed on degraded patch reefs in the southeast section, where it attained largest thallus sizes. Maybe something like this:
Our observations further show the persistence of Galaxaura on degraded reefs both over a 17-month period and for at least four years. However, our data do not resolve potential seasonal variations of Galaxaura during the cooler northeast monsoon season. In the tropical waters of Florida, U.S., Galaxaura shows seasonal persistence with no significant change in abundance among seasons [58].
The causes for Galaxaura blooms in the southeast area of the Dongsha lagoon are not understood. The dominance of macroalgae, such as Galaxaura, on degraded reefs is probably the consequence rather than the cause of initial coral decline [1,30,59,60]. There are several potential factors that could facilitate a Galaxaura outgrowth after a disturbance, including shallow, calm, and sheltered habitats, and nutrient-rich waters with high turbidity. Other members of the Galaxauraceae family, such as Tricleocarpa, are known to prefer sheltered habitats [61], and bloom under high water turbidity and low irradiance [62]. The southeast section of the lagoon, where Galaxaura is most abundant, is sheltered by the relatively wide reef flat (2 km), and characterized by shallow waters (1-5 meter) and low current [63]. Galaxaura was absent from environments with strong water currents, i.e. west lagoon, channels, and forereef, where strong, erosive currents [35,63] may create unfavorable conditions for Galaxaura to establish.
Due to limited water exchange with the open ocean, coral communities and associated fauna of the shallow, semi-closed lagoons are highly vulnerable to heat stress, eutrophication, and hypoxia [64-66], especially under the backdrop of climate change [67]. Recurrent mass mortality events in the Dongsha lagoon in 2014 and 2015 killed thousands of fish, cephalopods, gastropods, and crustaceans, and eradicated more than 25 km2 of seagrass, potentially caused by hypoxia [35,68]. During our 2017 survey we recorded extremely low densities of macroinvertebrates, including echinoids, sea cucumbers, lobsters, and giant clams (Table S4). Galaxaura filamentosa was among the very few macroalgae that proliferated in the Hikeru the lagoon of French Polynesia, following a hypoxia-induced mass mortality event [18]. Elevated nutrients from seagrass die offs in the southeast lagoon and a potentially higher resistance to hypoxia may be additional factors promoting blooms of Galaxaura.
A long-term bloom of Galaxaura is likely to have profound implications on the recovery potential of degraded reefs. The observations of this study further suggest that Galaxaura divaricata is potentially allelopathic in competition with corals. Allelopathic competition with corals has previously been demonstrated for Galaxaura filamentosa [12,14,15,27-29]. Extract of G. filamentosa were shown to inhibit the settlement of Acropora tenuis larvae [29]. The susceptibility of coral to algal overgrowth depends on the type of coral [1,59] and the coral colony form. Branching corals appear most vulnerable to algal overgrowth [69]. In the lagoon of Dongsha Atoll G. divaricata frequently grew in contact with massive and branching Porites, e.g., P. lobata, P. cylindrica, and P. solida. Previous studies in the tropical Pacific reported similar observations, where Porites, in particular Porites cylindrica, was frequently found in contact with the allelopathic Galaxaura filamentosa, suggesting that Porites may be relatively tolerant to Galaxaura allelopathic chemicals [15]. Acropora corals are fast growing and considered competitive against macroalgae overgrowth [59]. However, the majority of the thermal-susceptible Acroporid species were eradicated following reoccurring bleaching events since 1998 [36,57]. The coral community in the lagoon of Dongsha Atoll has since been dominated by more thermal tolerant genera, such as Porites, Echinopora, Pavona, and Turbinaria [51]. These coral genera are rather slow-growing, and thought to be less competitive to macroalgae overgrowth [17,59]. Unlike crustose coralline algae (CCA), Galaxaura does not stabilize the coral reef matrix, and a long-term bloom of Galaxaura is likely to exacerbate the flattening of the complex three-dimensional coral reef structure, which may have consequences for reef biodiversity, ecosystem functioning and associated services [64,70].
Insufficient grazing after disturbance can lead to the establishment and full outgrowth of macroalgae beyond their initial stages [30]. Galaxaura is known to be largely unpalatable for various herbivorous fishes due to its calcareous thallus and low nutritional content [23,25,26,71,72]. Once established, large macroalgae are less efficiently consumed by herbivorous fishes [30,73]. Local herbivorous fish population on Galaxaura-dominated reefs in the Dongsha lagoon may not be effective to prevent or reverse the space monopolization by Galaxaura [30,74]. It is suggested that Galaxaura can alter the chemical microclimate on degraded reefs with potential implications on fish behavior. Butterfly fishes and other corallivores avoid corals in close association with Galaxaura filamentosa, making it potentially difficult for these trophic guilds to find food [75,76]. Water soluble chemicals of Galaxaura rugosa negatively impact the predator risk assessment of damselfish by nullifying their perception of predator warn odors [11]. In summary, the long-standing canopies of Galaxaura are likely to hamper coral larvae recruitment through allelopathic recruitment inhibition [29], ultimately preventing coral reef recovery [1,15].
Galaxaura divaricata provides a suitable substrate to host a variety of macroalgae. Some of the identified macroalgae on Galaxaura are widely important in coral overgrowth in response to disturbances, and are known for their allelopathic inhibition coral larvae recruitment, e.g. Lobophora [77,78], Dictyota bartayresiana [79,80], and cyanobacteria [81,82]. Here, we firstly report that the yet undescribed species, Lobophora sp28 [49], third most abundant macroepiphyte on Galaxaura, was frequently observed to overgrow and kill corals, preferentially branching Porites spp. (primarily P. cylindrica) through epizoism (Figs 5 and S2). Similarly, Lobophora hederacea overgrows and kills corals in New Caledonia through epizoism [83].
Galaxaura is known to be of low preference to various herbivorous fishes due to its calcification [84,85] and low nutritional value [25,84,86,87]. Galaxaura was proposed to provide associational refuge to more palatable macroalgae [86]. For instance, rabbitfishes prefer Caulerpa and Dictyota [88,89], but avoid Galaxaura [23]. These epiphytic macroalgae thus could indirectly gain the benefit by growing between the branches of Galaxaura, as they are less likely to be spotted by herbivorous fish. Commensalistic interactions are important for the establishment of less common, nutrient-rich seaweeds that grow in association with common, nutrient-poor seaweeds [80]. Even if the common seaweeds lowered the growth rate of the less common ones (competition), evidence suggest that the benefits provided by macroalgae associations outweigh the drawbacks through competition [80,90]. Moreover, the microscopic filaments of Galaxaura may facilitate the attachment of macroalgae spores, while the calcium carbonate branches may provide structural support for other macroalgae. Considering that an increase in substrate availability can enhance the biomass of macroalgae on the reef [1], we hypothesize that, by providing a suitable and sheltered substrate for epiphytic macroalgae, Galaxaura may facilitate the diversity and abundance of macroalgae on degraded reefs. This study is merely observational and does not provide experimental evidence for the facilitation of macroalgae diversity and abundance by Galaxaura. However, the abovementioned hypotheses would be of great interest awaiting future validation.
Conclusions
Our observations illustrated that the allelopathic and unpalatable seaweed Galaxaura divaricata can become dominant on degraded reefs in shallow, sheltered environments. On degraded coral reefs, the dominant macroalgae have a profound impact on coral recruitment and coral recovery. Allelopathic and unpalatable macroalgae such as Galaxaura were proposed to perpetuate their dominance on degraded reefs by chemically inhibiting the process of coral recruitment. Thus, a long-term dominance by the allelopathic, nutrient-poor Galaxaura could provide a negative feedback, perpetuating reef degradation. In addition, we suggest that Galaxaura provides a suitable substrate for a variety of macroalgae, potentially facilitating macroalgae growth and abundance on degraded reefs. Several common macroepiphytes on Galaxaura have been proven to be allelopathic in competition with corals and inhibit coral larvae settlement. Thus, degraded coral reefs dominated by Galaxaura may fail to recover, and face a substantial decline in biodiversity of corals, fishes, and other associated fauna, which may have far-reaching effects on coral reef ecosystem functioning and services. Macroalgal assemblages, such as the Galaxaura-macroepiphyte system, warrant further investigation to better understand the interactions between macrophyte inhabitants and habitat forming seaweeds and their ecological implications. There are 439 listed coral reef atolls on earth; among them are 335 with open or semi-enclosed lagoons [91]. Atoll lagoons are highly productive and serve as valuable and nursery habitat for marine life; however, they are most vulnerable to the effects of climate change [18,66,67]. Results from our study can be informative for the management and conservation of lagoons and shallow, inshore coral reef ecosystems, especially for coral reefs in the South China Sea and the Pacific Ocean, where Galaxaura is a common seaweed.
Acknowledgements
The authors would like to thank our colleagues of the joint project: “Patterns of Resilience in Dongsha Atoll Coral Reefs” for their wonderful collaboration and great support throughout this study. We thank Keryea Soong, the staff of the Dongsha Atoll Research Station (DARS), and the Dongsha Marine National Park (DAN), Coastal Guard Administration, Ministry of Marine Affairs for the logistic support. We would like to thank Prof. George P. Lohmann and Cherng-Shyang Chang for assistance with benthic surveys, as well as Jie-Shuen Li, and Pin-Chen Chen for assistance with fieldwork and DNA barcoding.
References
- 1.↵
- 2.↵
- 3.↵
- 4.
- 5.
- 6.
- 7.
- 8.
- 9.↵
- 10.↵
- 11.↵
- 12.↵
- 13.↵
- 14.↵
- 15.↵
- 16.
- 17.↵
- 18.↵
- 19.↵
- 20.
- 21.↵
- 22.↵
- 23.↵
- 24.↵
- 25.↵
- 26.↵
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.↵
- 34.↵
- 35.↵
- 36.↵
- 37.↵
- 38.↵
- 39.↵
- 40.
- 41.↵
- 42.↵
- 43.↵
- 44.↵
- 45.↵
- 46.↵
- 47.↵
- 48.↵
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.↵
- 63.↵
- 64.↵
- 65.
- 66.↵
- 67.↵
- 68.↵
- 69.↵
- 70.↵
- 71.↵
- 72.↵
- 73.↵
- 74.↵
- 75.↵
- 76.↵
- 77.↵
- 78.↵
- 79.↵
- 80.↵
- 81.↵
- 82.↵
- 83.↵
- 84.↵
- 85.↵
- 86.↵
- 87.↵
- 88.↵
- 89.↵
- 90.↵
- 91.↵