**1. Introduction**

#### *1.1. Uncovering Taxonomic Progress*

The identification of scleractinian corals based solely on morphology is challenging because some scleractinian species can exhibit environment-correlated variations in morphology, i.e., Ecomorphs [1]. In addition, species display phenotypic plasticity across their distribution, making it difficult to rely on shared morphological features to identify them [2,3]. Therefore, it is important to combine morphological and molecular characteristics to improve the accuracy of the determination of evolutionary relationships.

The traditional classification of scleractinia into seven suborders was out of date [4–7]. Given the comprehensive study of the entire taxon with morphological and molecular approaches, the scleractinian corals can be generally divided into three major groups: basal, robust and complex [8]. Furthermore, they are separated into 21 clades (I-XXI) [8,9]. Many

**Citation:** Chen, C.-J.; Ho, Y.-Y.; Chang, C.-F. Re-Examination of the Phylogenetic Relationship among Merulinidae Subclades in Non-Reefal Coral Communities of Northeastern Taiwan. *Diversity* **2022**, *14*, 144. https://doi.org/10.3390/d14020144

Academic Editors: Michael Wink and Simone Montano

Received: 29 December 2021 Accepted: 14 February 2022 Published: 17 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**<sup>\*</sup>** Correspondence: b0044@email.ntou.edu.tw

scleractinian corals at family and genus were revised or remained unclear taxonomic position (Scleractinia *incertae sedis*). For example, *Diploastrea helipora* and *Montastraea carvernosa* were separated into Diploastraeidae Chevalier & Beauvais, 1987, and Montastraeaidae Yabe & Sugiyama, 1941, respectively [10]. Euphylliidae Milne Edward & Haime, 1857, contains six genera: *Ctenella* Matthai, 1982; *Euphyllia* Dana, 1846; *Galaxea* Oken, 1815; *Gyrosmilia* Milne Edwards & Haime, 1851; *Montigyra* Matthai, 1928; *Simplastrea* Umbgrove, 1939; and *Frimbriaphyllia* Veron & Pichon, 1980, the last of which was redefined from the conventional *Euphyllia ancora*, *E. yaeyamaensis*, and *E. divisa* [11]. In addition, the genera *Nemenzophyllia*, *Physogyra*, and *Plerogyra* were removed from Euphylliidae because they formed a separate clade with *Blastomussa* (clade XIV) [9,12].

#### *1.2. Revision of Merulinidae (Clade XVII)*

The species identification of Faviidae, Gregory, 1900 and Wells, 1956 was based on their budding patterns and macromorphological characteristics They were traditionally subdivided into two subfamilies based on whether their budding was primarily intracalicular (*Caulastraea*, *Favia*, *Diploria*, *Favites*, *Oulophyllia*, *Goniastrea*, *Platygyra*, *Leptoria*, *Hydnophora*, *Manicina*, and *Colpophyllia*) or extracalicular (*Montastraea*, *Diploastrea*, *Cyphastrea*, and *Echinopora*). A third, smaller, subfamily displays intracalicular budding and very well-developed septal lobes (trabecular versus lamellar, continuous versus discontinuous). Genera within the Faviinae are distinguished by having a colony form (ceroid versus plocoid, mendroid versus phaceloid) and the columella structure (trabecular versus lamellar versus continuous versus discontinuous).

Based on the molecular results, the genera of Faviidae not only displayed a polyphyletic pattern, but were also clustered together with species from four conventional coral families: Faviidae Milne Edwards & Haime, 1857; Merulinidae Verrill, 1995; Pectinidae Rafinesque, 1815; and Trachyphylliidae Well, 1956, which had previously been recovered as Merulinidae (XVII) [10,13–17]. The faviid corals outside of clade XVII were assigned to other families. For example: *Plesiastrea versipora*, *Diploastrea helipora*, and *Montastraea cavernosa* were reclassified as Plesiastreidae Dai & Horng, 2009, Diploastraeidae Chevalier & Beauvais, 1987, and Montastraeidae Yabe & Sugiyama, 1941, respectively. Faviidae is limited to Atlantic corals such as *Favia*, *Diploria*, and *Manicina* because they are evolutionarily divergent to the Pacific corals [18,19]. Furthermore, phylogenies based on multiple genetic markers and morphological characteristics demonstrated that the species/genera Merulinidae are divided into nine subclades (A, B, C, D/E, F, G, H, and I) [10,14,16]. *Paramontastraea*, *Orbicella*, and *Astrea* are new genera in the Merulinidae, revised from *Montastraea*. Given these results, Merulinidae contains the most genera (with 24) and the second-most species (with 149) among the scleractinians [20] (Supplementary Table S1). Its species are commonly distributed in the Indo-Pacific [2,21].

#### *1.3. Taiwan Taxonomy and Species Diversity*

Taiwan is located at the center of the Philippine–Japan Island arc at a latitude of 21.90◦ N to 25.3◦ N, crossing from the Tropic of Cancer close to the northern tip of the Coral Triangle [22]. To date, 317 scleractinian coral species have been reported in Taiwan and display a latitudinal gradient of decreasing species diversity from south to north [23,24]. In addition, coral assemblages contain 21 genera covering 87% of the total number of genera of merulinid corals and 89 species covering 60% of the total number of merulinid species [25]. Taxonomic phylogenetic studies of scleractinian corals collected from Taiwan are very limited [9,11,26–29]. In addition, biogeographical integration is needed on a larger scale. For example, *Polycyathus chaishanensis* (Caryophyllidae) was proposed to be endemic to Taiwan [27]. Later, this species was also found to inhabit Indonesia, based on molecular evidence [30]. *Euphyllia ancora* has been a model species for studies on sexual reproduction [31] and its genus was recently revised to *Fimbraphyllia* [11]. This revision created an important foundation on the convergen<sup>t</sup> and divergent functionalities

of genes and compared functional genes among the cnidarians underlaying precisely the phylogenetic position of the studied species.

#### *1.4. Purpose of This Research*

The phylogeny of Merulinidae reconstructed in Huang et al. [16] was based on samples/taxa from Australia, Singapore, Japan, and the Philipines in the Pacific Ocean and the Atlantic Ocean. Taiwan is located in the Pacific Ocean; it is an important stepping stone between the Philippines and Japan. Merulinid corals are major spawning members and consistently spawn between summer and fall in non-reefal coral communities in northern Taiwan [32]. These spawning corals are important for maintaining local recruitment, providing heterogenetic materials to the local population and connecting across different populations. However, some convergen<sup>t</sup> macro-morphological characteristics make it challenging to identify some genera in the field, such as *Goniastrea* (ceroid form), *Favites* (ceroid and plocoid forms), and *Diploastraea* (plocoid form) [24,33]. In addition, species identification for spawning corals based on morphological criteria in the fields and underwater photographs is difficult because the polyps are deformed when "the mature sperm and eggs move to the mouths of polys" (i.e., bundle setting).

As mentioned above, these challenges can be resolved by molecular approaches, as was demonstrated by Huang et al. [10]. For example, ceroid forms of *Goniastrea*, *Diploastraea*, and *Favites* are clearly separated in subclades A, B, and F based on phylogenetic reconstruction using multiple loci [10]. Therefore, Chen et al. [32] identified the species to the genus level of each specimen using molecular approaches and the BLAST tool [34]. Subsequently, specimens were identified to species level using the morphology of their skeletons. The established molecular database of merulinid corals can provide further insight into the phylogenetic relationships among the subclades of Merulinidae. The objectives in this present study were to: (1) establish a molecular database of spawning corals of Merulinidae from Taiwan, which have not been studied before; (2) re-examine the phylogenetic relationship between the specimens collected from northern Taiwan and the merulinid corals in previous studies, using phylogeny reconstructions based on multiple loci; and (3) record any new species or subclades we might find in this region.

#### **2. Materials and Methods**

#### *2.1. Sample Collection*

Chen et al. [32] demonstrated that the spawning season for merulinid corals is July to August, from 2014 to 2016, in northeast Taiwan. Merulinid corals with bundle-setting behavior and released bundles still attached outside of the mouths of polyps were collected at night by scuba diving. Some corals were collected at two offshore islands and their sexual reproductive behavior was observed using histological approaches [32]. A total of 65 specimens from four sites were chosen for this study: 26 specimens from Pitoujiiao (25◦0734 N, 121◦5455 E), 21 from Longdong (25◦0502 N, 121◦5509 E), 3 from Keelung Island (25◦0734 N, 121◦5455 E), and 12 from Kueishan Island (24◦8419 N, 121◦5706 E) (Figure 1). Coral fragments were collected by using chisels and hammers and separated into two parts. One was fixed in 90% ethanol for molecular analysis. The other was bleached in sodium hypochlorite until the tissue was entirely removed, rinsed in freshwater, and air-dried for the morphological analysis.

#### *2.2. Species Identification*

Chen et al. [32] identified 54 coral species in 23 genera and 8 families (Acroporidae, Agariciidae, Fungiidae, Lobophylliidae, Merulinidae, Poritidae, Pocilloporidae, and Psammocoridae), which were sexually reproductive between July and October. For Merulinidae, nine genera and 26 species collected from northeast Taiwan were chosen for the molecular phylogenetic study: *Astrea curta* (*n* = 5)*, Astrea annuligera* (*n* = 1), *Coelastrea aspera* (*n* = 2), *Coelastrea palauensis* (*n* = 1), *Cyphastrea chalcidicum* (*n* = 2), *Dipsastraea favus* (*n* = 4), *Dipsastraea lizardensis* (*n* = 1), *Dipsastraea matthaii* (*n* = 1), *Dipsastraea rotumana* (*n* = 1),

*Favites flexuosa* (*n* = 1), *Favites pentagona* (*n* = 7), *Favites stylifera* (*n* = 2), *Favites magnistellata* (*n* = 2), *Favites valenciennesi* (*n* = 2), *Mycedium elephantotus* (*n* = 1), *Mycedium robokaki* (*n* = 1), *Mycedium mancaoi* (*n* = 1), *Paragoniastraea australensis* (*n* = 5), *Paragoniastrea deformis* (*n* = 6), *Pectinia paeonia* (*n* = 1), *Pectinia lactuca* (*n* = 1), *Platygyra daedalea* (*n* = 1), *Platygyra lamellina* (*n* = 2), *Platygyra ryukyuensis* (*n* = 5), *Platygyra pini* (*n* = 2), *Platygyra sinensis* (*n* = 1), and *Platygyra verweyi* (*n* = 3). Those specimens were identified to the genus level using molecular sequences and BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 2 April 2020) [34]. Subsequently, individuals were identified to the species level using morphological keys, notably Dai and Cheng [25]. Specimens that could not be identified morphologically (cerioid corals: *Goniastrea* and *Favites*, plocoid corals: *Favites* and *Dipsastraea*, unknown species, etc.) were preliminarily identified to the genus level and then re-evaluated after molecular analyses. DNA extraction, PCR amplification, and sequencing

**Figure 1.** Map showing sampling sites at Pitoujiiao, Longdong, Keelung Island, and Kueishan Island in northeastern Taiwan.

Genomic DNA was extracted from 90% ethanol-preserved tissue specimens using the automated LabTurbo Nucleic Acid Mini Kit LGD480-220 (Taigen Bioscience Corporation), following the manufacturer's protocols. A total of four genes were amplified from the collected specimens, including one mitochondrial marker and three nuclear markers, following Huang et al. [16]: (1) cytochrome c oxidase subunit I segmen<sup>t</sup> (MCOIF: 5- TCTACAAATCATAAAGACATAGG-3, MCOIR:5-GAGAAATTATACCAAAACCAGG-3); (2) nuclear ribosomal internal transcribed spacer segmen<sup>t</sup> (ITS, A18S: 5-GATCGAACGGTTT AGTGAGG-3, ITS-4: 5-TCCTCCGCTTATTGATATGC-3); (3) two variable domain (D1 and D2) at 5end of 28S ribosomal RNA segmen<sup>t</sup> (C1: 5-ACCCGCTGAATTTAAGCAT-3, D2MAD: 5-GACGATCGATTTGCACGTCA-3); and (4) histone H3 segmen<sup>t</sup> (H3F: 5- ATGGCTCGTACCAAGCAGACVGC-3, H3R: 5-ATATCCTTR GGCATRATRGTGAC-3). PCR was carried out using 12.5 μL of Fast-RunTM Advanced Taq Master Mix (Protech, Taipei, Taiwan), 10 mM each of forward and reverse ITS primer, 10–100 ng/μ<sup>L</sup> DNA template, and deionized water to a final volume of 25 μL. The PCR profiles were as follows: an initial denaturation stage (95 ◦C, 5 min); 35 cycles of a denaturation step (95 ◦C, 30 s, an annealing step (54 ◦C, 40 s); an elongation step (72 ◦C, 7 min); and a final extension at 72 ◦C, for 5 min. The PCR products were confirmed by electrophoresis and subcloned into a pGEM-T easy vector (Promega, Madison, WI, USA). Three inserted cDNA fragments were sequenced

with the pUC/M13 forward and reverse primers using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Forster City, CA, USA).

#### *2.3. Sequence Management, Alignment, and Matrix*

The raw forward and reverse sequences were edited and assembled into consensus sequences by the CodonCode Aligner V6.0.2 program (CodonCode Corporation Dedham, MA, USA). To exclude sequences amplified from zooxanthellae, the consensus sequences obtained were used to perform the BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 2 April 2020) [34]. The sequences obtained from the collected specimens of spawning corals in northern Taiwan were deposited into the NCBI GenBank (accession numbers in Supplementary Table S1). Newly obtained sequences for COI (*n* = 58), ITS (*n* = 59), 28S (*n* = 63), and histone (*n* = 62) were combined with sequences retrieved from public sources (Table S1).

All sequences for each gene were automatically aligned with the accurate alignment option (E-INS-i) in MAFFT v.7 ([35]; http://mafft.cbrc.jp/alighment/server/, accessed on 10 January 2021) under default parameters. The resulting multiple sequence alignments were translated into inferred amino acid sequences as a guide for inferred gap placement between coding regions using Se-Al v.2.0a11 [36]. The amino acid residue and nucleotide were manually adjusted to minimize the gaps. PAUPRat software v.3.1 [37] on the CIPRES Science Gateway (http://www.phylo.org, accessed on 10 January 2021) [38] was used to calculate descriptive statistics (sequence variations and informative sites) for the compared sequences of each gene.

#### *2.4. Molecular Datasets*

Some sequences were not obtained because the gene failed to amplify during PCR. Operational taxonomic units (OTUs) were created for each gene for the phylogeny reconstruction. The phylogeny reconstructions were conducted based on the combined gene matrix (COI, 28S, ITS, and histone); IGR (noncoding intergenic region between COI and the formylmethionine transfer RNA gene) was ignored because the overall alignment was not similar enough.

The sequences of merulinid corals published in Huang et al. [16] were retrieved from GenBank. They included 19 genera in Merulinidae: *Merulina* (2 species), *Caulastraea* (3 species), *Cyphastrea* (3 species), *Dipsastraea* (13 species), *Echinopora* (5 species), *Favites* (13 species), *Goniastrea* (5 species), *Hydnophora* (2 species), *Leptoria* (2 species), *Mycedium* (2 species), *Orbicella* (1 species), *Oulophyllia* (2 species), *Pectinia* (3 species), *Platygyra* (8 species), *Scapophyllia* (1 species), and *Trachyphyllia* (1 species); two resurrected genera (*Astrea* (2 species), *Coelastrea* (2 species)); and one new genus (*Paramontastraea* (1 species)). In total, we retrieved 124 sequences for 28S rDNA, 121 sequences for histone H3, 91 for ITS rDNA, and 112 for COI from the GenBank.

The clades distant from the merulinid corals (XVIII-XXI) were included for the phylogenetic inference following Huang et al. [10]. These sequences were comprised of three species of Lobophylliidae (*Moseleya latistellata*, *Acanthastrea echinata*, and *Lobophyllia corymbosa*), three species of Faviidae (*Montastraea multipunctata*, *Favia fragum*, and *Mussa angulosa*), and one species of Plesiastreidae (*Plesiastrea versipora*). Phylogeny reconstructions were created for each gene, along with four combined datasets based on maximum likelihood and Bayesian analyses.

#### *2.5. Molecular Phylogenetic Analysis*

The maximum likelihood (ML) trees of each partition were reconstructed with raxml-GUI v.2.0 [39] using the best model (GTR+I+G). The five datasets, including three nuclear genes (ITS, 28S, and histone H3), one mitochondrial gene (COI), and a combined gene dataset, were partitioned based on coding position. The combined gene datasets were conducted with five independent runs, and the tree with the best ML scores was selected

as the final tree. Nodal support was assessed by bootstrapping, and only the nodes with ≥70 [40] based on 1000 pseudo-replicates were shown.

Bayesian inference (BI) was carried out in MrBayes v.3.2.6 [41]. PartitionFinder was used to select the best partition scheme and accompanying substitution model, according to the Bayesian information criterion [42]. The best-fit substitution model was determined by ProtTest3. Two Monte Carlo Markov chains (MCMCs) were run for 4 × 10<sup>6</sup> million generations in two simultaneous runs, each with four different chains. The convergence of the estimates was checked by the standard deviation of split frequencies and by monitoring the likelihood score over time using Tracer v.1.6 [43]. Trees were sampled every 1000 generations, with the first 2500 (25%) discarded as "burn-in." The remaining sampled trees were collected to construct a 50% majority-rule BI consensus tree. Nodal support from BI was assessed, and only nodes with ≥0.90 posterior probabilities (PPs) were shown.

The output trees were further edited by FigTree v1.3.1 [44]. *Plesiastrea versipora* (clade XIV, Plesiastreidae) was set as a distant outgroup to root the inferred trees. The subclades within Merulinidae (XVII) were divided into subclades A-I, following Huang et al. [10,16].
