The habitat-modifying red alga Ramicrusta on Pacific reefs: A new generic record for the Tropical Northwestern Pacific and the description of four new species from Guam

The genus Ramicrusta (order Peyssonneliales) is a new record for Micronesia, with range expansions of Ramicrusta fujiiana and R. lateralis to Guam. In addition, four species (Ramicrusta adjoulanensis, R. asanitensis, R. labtasiensis, and R. taogamensis) are newly described from Guam using molecular and anatomical characters. Ramicrusta lateralis specimens from Guam share most anatomical features with the holotype description from Vanuatu, but the plants from Guam are more tightly adherent, rigid, and robust than those of Vanuatu. Ramicrusta adjoulanensis possesses a well-developed epithallus with frequent cell fusions, secondary pit connections, and lacking hair bases or trichocytes, similar to Ramicrusta bonairensis. Ramicrusta adjoulanensis differs from other Ramicrusta species in having occasionally free margins and being attached by frequently produced, relatively long rhizoids (75–100 μm long). Ramicrusta asanitensis shares features with many other species, but the thickness of the crust (upwards of 2 mm thick), heavy calcification in the epithallus, and the extent of secondary, tertiary, and quaternary growth, differentiate it from other Ramicrusta species. Ramicrusta labtasiensis shares features with its close relative Ramicrusta lateralis but possesses frequent, robust, and relatively long rhizoids (75–95 μm long) throughout its entire undersurface. Ramicrusta taogamensis resembles its close relative Ramicrusta appressa but is primarily distinguished by its generally well-developed epithallus with occasional secondary pit connections and cell fusions. The six species reported here make Guam equal to Vanuatu in currently having the highest known species richness of Ramicrusta in the world.


Introduction
Among crustose calcifying red algae (CCRA), calcifying and encrusting members of the Peyssonneliales [1] have historically been overlooked in favor of the more frequently studied members of the Corallinophycidae. However, advances in molecular techniques have greatly other crustose algae (e.g., Peyssonneliales and Lobophora spp.) that can cover up to 29% of the benthic substrate [19]. R. bonairensis has also been observed overgrowing corals and sponges on disturbed Caribbean reefs [25,26]. Below, we describe four new species of Ramicrusta from Guam based on comparative genetic and morphological analyses. R. fujiiana and R. lateralis are also reported as new species records for Guam. These are the first records of the genus Ramicrusta for the Tropical Northwestern Pacific marine province [27].

Collection and morphological analysis
Samples were collected by reef wading, snorkeling, and diving at various sites around Guam (Fig 1). Collection permits were obtained from the Guam Department of Agriculture's Division of Aquatic and Wildlife Resources (DAWR). Specimens were photographed in situ, collected, and photographed again before being transferred to holding tanks with running seawater until DNA extraction. Portions of samples were preserved in formalin, silica gel, and air-dried as herbarium specimens. Specimens were deposited at the University of Guam Herbarium (GUAM). However, the Ramicrusta fujiiana specimen that was collected, photographed, and sequenced was lost from the holding tank before it could be preserved. As such, the report of R. fujiiana for Guam is based on the DNA sequence data obtained from the specimen before it was lost. For anatomical observations, material was hand-sectioned using a razor blade and embedded on 12.7 mm pin mounts using colloidal graphite with isopropanol base (Energy Beam Sciences). The sections were sputter coated using an Emitech SC7620 Sputter Coater (Quorum Technologies Ltd., Laughton, East Sussex, United Kingdom). Anatomical observations were made and imaged using a Phenom G2 Pro desktop scanning electron microscope (Phenom-World B.V., Eindhoven, The Netherlands).

Molecular analysis
For molecular analyses, total genomic DNA was extracted using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, CA) or the GenCatch Blood & Tissue Genomic Mini Prep Kit (Epoch Life Science Inc., Missouri City, TX) following the manufacturers' bench protocol. The mitochondrial COI-5P was polymerase chain reaction (PCR) amplified using a newly designed forward primer TS_COI_F01_10 (5'-TCGARTCYCGTCTCTCTCG -3') and the reverse primer GWSRx [28] following the amplification profile 95˚C for 3 minutes; 35 cycles of 94˚C for 40 seconds, annealing at 48˚C for 40 seconds, extension at 72˚C for 100 seconds; a final extension at 72˚C for 10 minutes. Chloroplast psbA was amplified using the primers developed by Yoon et al. [29] following the amplification profile 95˚C for 3 minutes; 35 cycles of 94˚C for 40 seconds, annealing at 50˚C for 40 seconds, extension at 72˚C for 100 seconds; a final extension at 72˚C for 10 minutes. Plastid rbcL was amplified using the forward primer F57 [30] and the reverse primer rbcLrevNEW [31] following the amplification profile reported by Saunders & Moore [31]. PCR products were sent to Macrogen Inc. (Seoul, Republic of Korea) for purification and DNA sequencing.
Alignments for each of the gene regions were created using the MUSCLE plugin [32] in Geneious Pro 11.0.5 [33]. The COI-5P, rbcL, and psbA alignments were all analyzed independently prior to a combined analysis of all three genes. An alignment of fifty-one homologous COI-5P sequences was used to establish the phylogenetic relationship of Ramicrusta species from Guam and all but two currently described species of the genus. Lack of available sequence data excluded Ramicrusta calcea from phylogenetic analyses, while Ramicrusta melanoidea was excluded because of its high average COI-5P sequence divergence with other Ramicrusta Map of Guam identifying the sites from which Ramicrusta specimens were collected. Type localities of the four species being described are in red or pink. Scale bar = 20 km. (3)(4) Aerial photographs depicting the type localities of R. labtasiensis (Point # B) and R. taogamensis (Point # C). (5-7) Aerial photographs showing the type localities of R. adjoulanensis (Point # G) and R. asanitensis (Point # F). Maps were created using the ArcGIS computer software (Esri, Redlands, CA), and aerial photographs were captured on-site using a drone.
https://doi.org/10.1371/journal.pone.0259336.g001 species. Individual analyses of rbcL and psbA were limited by a lack of sequences available for comparison. A combined analysis of all three genes was used to establish phylogenetic relationships within the genus Ramicrusta. 51 new DNA sequences were generated for Ramicrusta specimens from Guam, of which 24 COI (MW960726-MW960749), 8 rbcL (MW960750-MW960757), and 19 psbA sequences (MW960758-MW960776; S1 Table). For all alignments, the general time reversal + invariable sites + gamma distribution (GTR+I+G) evolutionary model was selected as the optimal model using jModeltest 2.1.3 [34]. The concatenated alignment used in the combined analysis was partitioned by gene, and GTR+I+G was selected as the optimal model for each partition in the alignment. Phylogenetic analyses were performed for all alignments using maximum likelihood (ML) methods in RAxML [35]. The proportion of invariable sites and gamma shape parameters were estimated from the data, and sequence divergence was calculated using the MEGA version X computer software [36]. Sequence divergence was calculated using a neighbor-joining algorithm under a Kimura 2-parameter substitution model, which has been most often used when describing or reporting new Ramicrusta species [13,19,23]. Nonparametric bootstrapping (1000 replicates) was used to estimate node support. Bayesian inference was completed for each alignment using the MrBayes 3.1.2 [37] plugin in Geneious Pro 11.0.5 [32]. Each alignment was run for 1,000,000 generations with trees sampled every 100 generations, and the first 3,000 trees were discarded as burn-in (average standard deviation of split frequencies < 0.01). All COI-5P, rbcL, and psbA sequences obtained were deposited in GenBank (S1 Table), and once released, all COI-5P and rbcL sequences will also be available in the Barcode of Life Database (BOLD) [38].

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLoS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to World Register of Marine Species (WoRMS), from where they will be made available to the Global Names Index. The WoRMS LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http:// marinespecies.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.

Molecular and phylogenetic results
Phylogenetic analyses of the official barcode marker for red algae, COI-5P, supported the recognition of four new Ramicrusta species from Guam (Fig 2). RbcL and psbA phylogenies also support the recognition of four new Ramicrusta species, but the lack of sequences for previously described species does not allow for a comprehensive evaluation of phylogenetic relationships (S1 and S2 Figs). Analysis of the partitioned COI-5P, rbcL, and psbA alignment was congruent with the most taxon-complete COI-5P analysis (Fig 3).  Thallus was orangish-purple, completely calcified, closely appressed, and was tightly adherent to the substratum (Fig 4). The habit of the Guam specimen differed from the reddishorange specimens from Brazil, but R. fujiiana specimens from Guam and Brazil shared their strong adherence to the substratum [22]. The Guam specimen was unfortunately lost from the holding tank before further anatomical observations could be completed, so the report of R. fujiiana for Guam is based on DNA sequence data. The COI-5P barcode sequences of Guam

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sample was nearly identical, with a maximum 0.36% and average 0.08% intraspecific sequence divergence to the R. fujiiana specimens from Brazil. Phylogenetic analyses supported the report of R. fujiiana for Guam based on DNA sequence data (Figs 2 and 3). The difference in environment between the sampling locations in Guam and Brazil could explain the differences in habit between these genetically equivalent plants. Thalli were brown to reddish brown and heavily calcified ( Fig 5). Crusts were 225-550 μm thick and closely appressed. Typically, crusts were tightly adherent but loosely attached around some of the margins (Fig 5). Hypothallial filaments were parallel and composed of dorsally inflated oval cells that gave rise to assurgent perithallial filaments at broad angles. Plants were attached by squat, robust, thick-walled unicellular rhizoids (c. 50 μm long, 12-16 μm wide), which originated from the ventral portion of hypothallial cells and penetrated the thick (15-25 μm) hypobasal cuticle ( Fig 5). Perithallial filaments were simple or occasionally irregularly branched. Portions of secondary growth as well as overgrowth were present. Secondary growth could be recognized as alternating stacked layers of epithalli and lower perithalli (absence of hypothallial layers), while overgrowth appeared as two fully formed thalli stacked atop one another. Cells of the lower perithallus were thick walled, heavily calcified, and were frequently connected to adjacent cells via fusion or secondary pit connections ( Fig 5). The epithallus was thin, lacked secondary pit connections and cell fusions, and was composed of three to four tiers of small rectilinear cells (Fig 5). Hair bases or trichocytes embedded in the upper perithallus were large (c. 20 μm long and c. 23 μm wide), bullet-shaped, and terminated filaments of three or four cells (Fig 5). Reproduction was not observed.
The COI-5P barcode sequences of the four Guam samples were nearly identical (average 0.08% intraspecific sequence divergence) to that of the holotype of R. lateralis. They also shared anatomical features such as the structure of the epithallus, perithallial filaments being borne from the hypothallus at broad angles and having portions of secondary growth. There were, however, differences in their gross morphologies: the Guam specimens were tightly adherent to the substrate throughout, while only free around some of the margins. Crusts of the Guam plants were typically thinner, but they were rigid and robust, as opposed to being brittle in Vanuatu [13]. The difference in environment between the sampling locations in

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Guam and Vanuatu could explain the morphological differences between these genetically equivalent plants. Thalli were burnt orange to burgundy, tightly adherent, and irregularly lumpy due to irregularities in the substrata (Fig 6). Crusts were calcified throughout and typically closely appressed to the substrate but occasionally free at the margins. Crusts were relatively thick, typically reaching 350-600 μm in thickness. Hypothallial cells were parallel and composed of dorsally inflated oval cells that centrally gave rise to assurgent perithallial filaments. Rhizoids were frequently produced, unicellular, and were 75-100 μm long and 10-14 μm wide (Fig 6). Rhizoids were cut off from the ventral portions of hypothallial cells and emerged from the thick (typically 30-35 μm thick) hypobasal cuticle. Cells of the lower perithallus were also oval, but less dorsally inflated than the hypothallial filaments. Cells of the lower perithallus are typically large (18-32 μm high and 16-22 μm wide) and are connected to adjacent cells commonly by pit connections and occasionally by cell fusions (Fig 6). Cells in the mid-perithallus rapidly decrease in size, similar to what was observed in R. bonairensis [15]. The epithallus is generally well developed, often comprising at least half of the entire perithallus (Fig 6). Upper perithallial cells were commonly connected by secondary pit connections or fused with adjacent cells. Hair bases or trichocytes were absent in the upper perithallus and the upper perithallial filaments were crowded due to occasional branching in the upper perithallus (Fig 6). Reproductive features were not observed.
Ramicrusta adjoulanensis shares morphological characteristics with its close relative R. bonairensis, such as the significant decrease in cell size in the mid-perithallus, the well-developed epithallus with frequent cell fusions and secondary pit connections, and the lack of hair bases or trichocytes. Ramicrusta adjoulanensis was distinguished from R. bonairensis primarily by its attachment, with crusts that were occasionally free at the margins and by its relatively long rhizoids (75-100 μm long) that were frequently produced and penetrated the thick hypobasal cuticle. These features combined with DNA sequence divergences differentiated Ramicrusta adjoulanensis from R. bonairensis and other species of the genus. Thalli were dark maroon, heavily calcified, and formed closely appressed and tightly adherent crusts on various secondary reef structures such as large rocks and dead corals (Fig 7). The thallus surface contained small, rounded outgrowths, and the crusts were significantly thicker (upwards of 2 mm, but typically 500-1000 μm) than most other Ramicrusta species (Fig 7). The hypothallial filaments were parallel and composed of elongate, distally inflated rhomboid to rectilinear cells that gave rise to assurgent perithallial filaments centrally or at variable angles (> 45˚). Plants were attached by short (50-80 μm) unicellular rhizoids that were cut off at the distal ventral corners of hypothallial cells and penetrated the thin (10-15 μm thick) hypobasal cuticle (Fig 7). Perithallial filaments were simple, and the perithallus was composed of distinct upper and lower zones. Portions of secondary to tertiary growth as well as overgrowth were present (Fig 7). Secondary and tertiary growth appeared as stacked layers of epithalli and lower perithalli, while overgrowth appeared as one fully formed crust growing atop another. Cells in the lower perithallus were large (15-30 μm long and 12-22 μm wide), distally inflated, and rectilinear to ovoid in shape. These cells were thick walled and heavily calcified, and displayed frequent lateral secondary pit connections or cell fusions (Fig 7). The epithallus was relatively thin and consisted of four to five cell tiers that lacked cell fusions and secondary pit connections. The cells were smaller than those in the lower perithallus but were still thick-walled and heavily calcified (Fig 7). Hair bases or trichocytes were large (20-24 μm long and 11-14 μm wide), bullet-shaped, heavily calcified, and terminated four to five-celled filaments (Fig 7). Hair bases or trichocytes were often, but not always, associated with a pore on the thallus surface (Fig 7). Reproductive features were not observed.
Ramicrusta asanitensis possessed certain features similar to those commonly found in other Ramicrusta species, namely a closely appressed habit and frequent secondary pit connections and cell fusions in the lower perithallus. In addition, it also possessed the thin epithallus shared with its close relatives R. appressa and R. fujiiana. However, Ramicrusta asanitensis differed from other Ramicrusta species by the heavy calcification in the epithallus (as well as elsewhere in the crust), the thickness of the crust (upwards of 2 mm thick), and the extent of its secondary, tertiary, and quaternary perithallial growth. These features, in conjunction with its distinct genetic sequences, distinguished Ramicrusta asanitensis from other Ramicrusta species.   . Ramicrusta labtasiensis (Fig 8: GH0015097; Fig 8: GH0015717). (32) In-situ image of the holotype specimen displaying one distinct coloration. Scale bar = 2 cm. Observed thalli expressed two distinct colorations. Thalli were either reddish brown to maroon, with patches of lighter brown scattered throughout, or burnt orange to maroon (Fig  8). Plants were brittle, closely appressed and tightly adherent to dead coral substrate or other calcifying red algae, and formed crusts that were 240-500 μm thick. Hypothallial filaments were parallel and composed of dorsally inflated cells that gave rise to assurgent perithallial filaments centrally or at broad angles. Plants were frequently attached by unicellular rhizoids (75-95 μm long and 10-14 μm wide) that cut off the distal ventral portion of hypothallial cells and penetrated the thin (12-15 μm) hypobasal cuticle (Fig 8). Cells of the lower perithallus were rounded, generally slightly elongate, and formed perithallial filaments that were often irregularly branched. Cells were heavily calcified and were frequently connected to adjacent cells via secondary pit connections or cell fusions (Fig 8). The epithallus was thin and was composed of two to four tiers of small rectilinear cells that were occasionally connected to cells of adjacent filaments via cell fusions or secondary pit connections. Pairs of upper perithallial filaments were often borne from the same cell in the mid-perithallus, resulting in filament crowding in the epithallus (Fig 8). Hair bases were infrequent, but often observed in close proximity to one another. Hair bases were bullet shaped, 23-27 μm long and 14-19 μm wide, and terminated filaments of three to four cells that were typically, but not always, associated with a pore on the thallus surface (Fig 8). Reproductive features were not observed.
Ramicrusta labtasiensis shared features with its close relative R. lateralis such as frequent cell fusions and irregular branching of filaments in the lower perithallus. Ramicrusta labtasiensis was primarily distinguished from R. lateralis by its attachment, where the thallus was frequently attached by robust, relatively long rhizoids (75-95 μm long) throughout its entire undersurface. It is also distinguished by the frequent branching and occasional secondary pit connections and cell fusions in the relatively thin epithallus. These features in combination with the distinct DNA sequences differentiate Ramicrusta labtasiensis from other Ramicrusta species. Thalli were deep red to crimson, heavily calcified, and formed tightly adherent and closely appressed crusts (250-500 μm thick) on bedrock (Fig 9). The thallus surface mimicked that of the substratum. The hypothallial filaments were parallel and composed of dorsally inflated, elongate rhomboid and rectilinear cells that gave rise to assurgent perithallial filaments centrally or at broad angles. Plants were attached by short (c. 70 μm long and c. 16 μm wide) unicellular rhizoids that terminated at the distal ventral portion of hypothallial cells and penetrated the relatively thick (~20 μm) hypobasal cuticle (Fig 9). The perithallus was

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composed of distinct upper and lower zones, divided by a horizontal linear series of cells that were irregularly shaped and frequently fused with the cells of neighboring filaments. The lower perithallus was largely composed of slightly dorsally elongate, thick-walled, and heavily calcified ovoid cells with frequent cell fusions and secondary pit connections (Fig 9). The upper perithallus (epithallus) was generally well-developed, comprising up to half of the entire perithallus. The upper perithallial cells were smaller and also slightly dorsally ovoid, forming a dorsal cortical layer of typically four to six cells thick. The upper perithallial cells were occasionally connected to adjacent cells by secondary pit connections or cell fusions (Fig 9). Hair bases were 17-22 μm long and 13-16 μm wide, circular to bullet-shaped, and terminated mostly three-to four-celled hair filaments (Fig 9). Reproduction was not observed.
The molecular results suggested that R. taogamensis was a cryptic sister-species of R. appressa. Ramicrusta taogamensis had much in common with its close relative R. appressa, such as its frequent cell fusions in the lower perithallus and tight adherence by short rhizoids, but was primarily distinguished by its generally well-developed epithallus with occasional cell fusions and secondary pit connections. The epithallus with distinct horizontal linear series of cells and the thicker hypobasal cuticle are vegetative features that are not collectively shared by any other Ramicrusta species. The differences in vegetative anatomy in combination with phylogenetic data distinguish Ramicrusta taogamensis from R. appressa and other Ramicrusta species.

Discussion and conclusions
Dixon and Saunders [13] used 4% K2P COI-5P interspecific variation as a threshold to distinguish Ramicrusta species. Most Ramicrusta species have been reported to exhibit low (< 1.0%) intraspecific sequence divergence values [13,15,19], with Ramicrusta appressa K.R.Dixon being the sole exception [13]. There was an average of 11.97% COI-5P sequence divergence between Ramicrusta species, ranging from 10.24% (R. appressa) to 13.99% (R. australica) divergence. For these analyses, Ramicrusta lehuensis was considered as a sister species to R. fujiiana in recognition of the diagnostic morphological features used to describe R. lehuensis despite the high COI-5P similarity between both species (< 2% divergence). An in-depth examination of the relationship between both species warrants further study. COI-5P sequences of R. textilis from Jamaica, Vanuatu, and Taiwan are nearly identical, while R. lateralis specimens from Guam and Vanuatu only demonstrate an average intraspecific divergence of 0.08% and R. fujiiana specimens from Guam and Brazil exhibit an average intraspecific divergence of 0.18%. The high sequence similarity within species from distant geographical regions further supports the recognition of four new Ramicrusta species from Guam. Specimens of R. appressa from Australia and the Philippines were 2.04% divergent from the holotype specimen from Vanuatu, leading Dixon and Saunders [13] to conclude that they may represent cryptic sister species. The description of Ramicrusta taogamensis renders R. appressa paraphyletic and thus provides support to recognize the R. appressa samples from Australia and the Philippines as a distinct, monophyletic species. Average sequence divergence between Ramicrusta taogamensis and the holotype specimen of R. appressa was 2.42%, supporting its taxonomic recognition as a new species. Apart from R. taogamensis, each new species was separated from its nearest-neighbor by more than 4.9% COI-5P barcode divergence. Low mean intraspecific barcode divergence (0.18% in R. fujiiana, 0.08% in R. lateralis, 0.33% in R. adjoulanensis, 0.00% in R. asanitensis, 0.14% in R. labtasiensis, and 0.11% in R. taogamensis) was also demonstrated for each species with more than one specimen sequences. The new Ramicrusta species exhibited 12.34% (R. adjoulanensis), 12.12% (R. asanitensis), 12.87% (R. labtasiensis), and 10.78% (R. taogamensis) divergence when compared to other sequenced representatives of the genus (Table 1). COI-5P sequences of R. textilis from Jamaica, Vanuatu, and Taiwan are nearly identical, while R. lateralis specimens from Guam and Vanuatu only demonstrate an average of 0.16% intraspecific divergence and R. fujiiana specimens from Guam and Brazil exhibit 0.36% average intraspecific divergence. Such high sequence similarity within species from distant geographical regions further supports the recognition of four new Ramicrusta species from Guam.
Crustose calcifying red algae (CCRA) have historically been difficult to identify, largely due to the cryptic diversity and morphological convergence among species [39,40], as well as their tendency to demonstrate phenotypic plasticity influenced by different environmental factors [41]. As such, studies of CCRA systematics have benefitted greatly from combining molecular methods and anatomical observations [3,13,15,27,38,41,42]. Twenty-four collections from Guam matched the anatomy and morphology of the peyssonnelioid red alga Ramicrusta. Anatomical observations paired with DNA sequence analysis revealed the presence of six Ramicrusta species. Two of these species corresponded to the previously described species, R. lateralis and R. fujiiana, the latter being confirmed by only DNA sequence analysis. Despite its relative abundance on many reefs on Guam, Ramicrusta was not known from Guam or anywhere else in the Tropical Northwestern Pacific marine province [27] until now. The COI-5P barcode is widely used to delimit species by employing the barcode gap, and it has been crucial in resolving species boundaries within Ramicrusta [13,15,19,22]. Ramicrusta adjoulanensis, R. asanitensis, R. labtasiensis, and R. taogamensis exhibited sufficient levels of interspecific divergence to be considered as new species within the genus Ramicrusta. The high relative abundance of Ramicrusta on certain reefs in Guam may be explained by an increase in disturbance events and an overall decline in reef health over the last decades [43]. Many of the Ramicrusta species in Guam were found on reef flats that experience severe fluctuations in temperature, salinity, and nutrients whereas others occurred abundantly on reefs that have been impacted by coral bleaching events or are chronically exposed to pulses of terrestrial runoff. Ramicrusta taxa have previously been reported to thrive in disturbed or environmentally stressed reef habitats [14,15,26]. There have not been many studies of crustose algae around Guam, and the diversity and ecology of Guam's CCRA communities are still poorly understood. Ramicrusta species have been reported in tropical to temperate waters across the globe. However, because of this study, which was based on a modest sampling effort, Guam now joins Vanuatu in having the highest reported Ramicrusta species richness of all marine ecoregions in the world (Fig 10). The recently documented high species richness of these small island nations suggests that the occurrence of Ramicrusta species is likely to be severely underreported globally. The potentially significant ecological impacts of Ramicrusta outbreaks on reef health [14,19,25,26] emphasizes the need for further investigations on Ramicrusta and CCRA diversity and ecology on a global scale.  Table. Species and sources of sequences used in the phylogenetic analyses.

Supporting information
(DOCX) Fig 10. A map of reported Ramicrusta species richness by marine ecoregion. The map, created using the ArcGIS computer software, includes all reported Ramicrusta species with available DNA sequence data. As with the molecular analyses, Ramicrusta calcea was excluded from the map due to its uncertain distribution range and lack of available sequence data. https://doi.org/10.1371/journal.pone.0259336.g010