**4. Discussion**

This is the first study to establish a molecular database for spawning corals, an important contribution to our understanding of genetic diversity in coral communities. We sequenced 1 species from Keelung Island, 9 from Kueishan Island, 13 from Longdong, and 12 from Pitoujiiao. In total, we sequenced 9 genera and 28 species, and most subclades were consistent with those of previous studies.

#### *4.1. Phylogentic Relationship of Merulinid Subclades*

The Merulinidae are defined as monophyletic in this study, confirming previous findings [10,16]. Of four Atlantic species, *Favia fragum*, *Mussa angulosa*, *Orbicella annularis*, and *Montastraea cavernosa*, only *O. annularis* is nested within the subclade C and a sister to *Cyphastrea* spp. The genus *Paramontastraea* Huang et al. 2014 [10] examined in this study was also a sister to *Echinopora* Lamarck, 1816, and nested within subclade I.

Increasing the sequence lengths, taxon sampling, and sampling locations may improve the phylogenetic relationship among taxa. Adding new sequences of merulinid corals from Taiwan generated longer aligned sequences with which to examine the phylogenetic relationships among the subclades of Merulinidae; as a result, most subclades changed their phylogenetic positions (Figure S1). For example, the tree topology reconstructed in Huang et al. [10] showed that *Hydnophora* (subclade H) is closer to *Favites* (subclade F). However, our reconstructed phylogenetic tree showed that the *Hydnophora* lineages only closer to *Favites pentagona* and the rest of *Favites* spp. are close to novel subclade K, which comprises *Astrea curta* and *Favites russelli*. In addition, subclade B shifted its position from D/E clades to subclade H and *Favites pentagona*.

As mentioned in Huang et al. [10,15,16], *Favites pentagona* and *Paragoniastrea autralensis* displayed polyphyletic patterns that require further investigation [10]. *Paragoniastrea australensis* was far from subclade A (*Goniastrea* spp.) and was clustered together with *Astrea curta*, *Astrea annuligera, Astrea devatieri*, and *Favites russelli* as a novel clade. *Favites pentagona* renders *Favites* polyphyletic in the molecular phylogeny and sister to the *Favites* spp. (subclade F) and subclade D/E [10]. Therefore, Huang et al. [10,16] suggested that these two species require further study with increasing sample collection from other locations. According to our new analysis, the molecular phylogenetic tree implied that *P. australensis* displayed a polyphyletic pattern, which is consistent with Huang et al. [10,16]. However, they were close to subclade A (*Goniastrea* spp.) and formed a novel subclade J (*Paragoniastrea* spp.). *Favites pentagona* formed a monophyletic pattern, which is different from Huang et al. [10,16]. They are separate from the major *Favites* spp. (subclade F) and are close to subclade H (*Hydnophora*).

#### *4.2. Application of Molecular Phylogentic Approaches*

Merulinidae corals with plocoid and ceroid forms are difficult to accurately identify to the genus level in the field because of their macro-morphological homoplasies [24,33]. In our phylogenetic analysis, all of the corals in the plocoid form were placed into subclades B, C, F, I, and/or K, including 18 *Dipsastraea* spp. (subclade B), 2 *Favites* spp. (B and F), 5 *Cyphastrea* spp. (C), and 2 *Astrea* spp. (B and K). Merulinidae corals in the ceroid form included 6 *Goniastrea* spp. (subclade A), 14 *Favites* spp. (F), 2 *Coelastrea* spp. (B), and 9 *Platygyra* spp. (G). Most of the samples we examined were either *Coelastrea* spp., *Favites pentagona*, *Platygyra* spp., *Paragoniastrea australensis*, or *Paragoniastrea deformis*. The genetic divergence between these four groups (subclades B, F, G, and J) may be driven by the differences in their sexually reproductive timing. *Favites pentagona* and *P. deformis* spawn in July, while *Platygyra* spp. and *P. australensis* spawn in August [32].

#### *4.3. Sexual Reproduction in Merulinidae*

Scleractinian corals have a complex sexual reproduction system, with the same species displaying different sexual reproduction patterns based on their geographic distribution [45]. The systematic pattern in the reproductive biology of Merulinidae, a hermaphroditic spawner, is highly conserved [45–47]. In this study, *Coelastrea aspera* collected from northern Taiwan was identified as a hermaphroditic spawner and placed in subclade B with the Singapore *Coelastrea aspera* [32,48]. *Coelastrea aspera*, from the Great Barrier Reef, is a spawner [49–52], whereas the nonspecific populations distributed in Palau are brooders [53,54]. The hermaphroditic *Coelastrea aspera*, from Okinawa, performs as a spawner [55] and a brooder [56–58]. A similar example, *Pocillopora damicornis*, is a brooder in most locations, but a spawner in western Australia [59–62].
