Microsatellite markers for multiple Pocillopora genetic lineages offer new insights about coral populations

Population genetics of the coral genus Pocillopora have been more intensively studied than those of any other reef-building taxon. However, recent investigations have revealed that the current morphological classification is inadequate to represent genetic lineages. In this study, we isolated and characterized novel microsatellite loci from morphological Pocillopora meandrina (Type 1) and Pocillopora acuta (Type 5). Furthermore, we characterized previously reported microsatellite loci. A total of 27 loci (13 novel loci) proved useful for population genetic analyses at two sites in the Ryukyu Archipelago, in the northwestern Pacific. Clonal diversity differed in each genetic lineage. Genetic structure suggested by microsatellites corresponded to clusters in a phylogenetic tree constructed from a mitochondrial open reading frame (mtORF). In addition, we found an unknown mitochondrial haplotype of this mtORF. These microsatellite loci will be useful for studies of connectivity and genetic diversity of Pocillopora populations, and will also support coral reef conservation.

microsatellites have been already reported in Pocillopora, we developed additional markers to widen the choice of markers for population genetics analyses between and within lineages. We developed 13 additional microsatellite markers from morphological Pocillopora meandrina and Pocillopora acuta colonies. In addition, we validated and characterized microsatellites previously reported from Pocillopora for populations in the Ryukyu Archipelago. Those were developed by Magalon et al. 20 , Magalon et al. unpublished, and Starger et al. 21 and recently, more loci have been isolated by Pinzón & LaJeunesse 13 and Torda et al. 8 . Gorospe & Karl 22 re-designed primers for two loci, PV7 from Magalon et al. 20 and Pd3-010 from Starger et al. 21 and designated them as Pd2-AB79 and Pd3-EF65, respectively. Using all of these validated microsatellite markers, we genetically characterized populations from multiple lineages of Pocillopora from Ueno and Yoshino, Miyako Island, in the Ryukyu Archipelago.

Results and Discussion
We successfully merged single sequences (2,523,457 reads comprising 1,034,311,164 bp for P. meandrina and 7,021,083 reads comprising 2,056,539,596 bp for P. acuta) from sequence data derived from genomic DNA. These were used for microsatellite detection and primer design. MISA and Primer3 were employed to identify 1,597 reads with primer sequences, including repeats under the following conditions (4 mer: 10 repeats or more; 5 mer: 8 repeats or more). After removing incomplete or extremely long repeats to facilitate fragment analysis, 752 reads remained (4 mer: 10 to 15 repeats; 5 mer: 8 to 12 repeats). Of these reads, we selected 50 with homologous repeat sequences and flanking regions, from P. meandrina and P. acuta, to be employed as cross-lineage microsatellite loci. Based on the genetic delimitation and definition by Pinzón et al. 10 , these morphological colonies displayed Type 1 and Type 5 mitochondrial open reading frames (mtORF), respectively. The nuclear ribosomal internal transcribed spacer 2 (ITS2) sequence from the Type 1 colony was ITS2 type T (see below).
We confirmed that 13 non-overlapping loci could produce sufficient PCR amplification in all genetic lineages, based on mitochondrial haplotypes derived from fragment analyses (Table 1). In our analyses using previously developed microsatellites, we could not confirm the proper amplification for genotyping of PV3 (Magalon et al. unpublished), PV5, PV6 20 , Pd3-002, Pd3-010 21 , or Pd13 8 , which have been widely used in other population genetics studies. However, redesigned primer sequences targeting flanking regions identified in databases, such as Gorospe & Karl 22 may have improved amplification of the loci, PV7 and Pd3-010. Actually, Pd3-010 was not useful, but Pd3-EF65 by virtue of nested primer sets, was useful even though it targeted the same locus as Pd3-010. Previously reported loci appear to be effective for populations in the Ryukyu Archipelago (Table 2). Clonal replicates were detected at both sites in Type 5 (Table 3). In addition, the population size of Type 5, based on genetic analysis appears to be large in Miyako Island, as in the Yaeyama Islands, where P. acuta exists abundantly 23 . Mean values of genetic indices across loci for each lineage/site with large numbers of multilocus genotypes (Types 1, 3, 5 at Ueno, Type 5 at Yoshino) are shown in Table 3. Detailed genetic indices for each locus are in Supplementary Table S1. The P ID ranged from 3.3e -26 to 1.5e -20 (Table 3); therefore, tandem use of these loci allows identification of genets with high resolution. Linkage disequilibrium was not significant (p > 0.05) in any microsatellite locus pairs after removal of replicate loci (PV7 is the same locus as Pd2-AB79). Novel microsatellite loci developed here are derived from the nuclear region since it exhibits only moderate heterozygosity and one or two peaks in  fragment analysis, excluding scoring errors. Also, polymorphism of zooxanthella loci was confirmed in the same multilocus lineage (MLL). These loci will facilitate combining multiple loci for effective multiplex PCR. Mitochondrial data showed two haplotype groups (Types 3 and 5) at the Ueno site, and haplotype groups of mtORF (Types 1, 3, 4, 5, 8, and unknown) at the Yoshino site. Furthermore, Type 1 was separated into two haplotype groups based upon ITS2. At least six lineages of Pocillopora inhabit the northwestern Pacific 12 . This study confirmed that all of these, plus one more, occur in the Ryukyu Archipelago. One unknown haplotype sequence was related to a sequence from Pocillopora brevicornis (GenBank accession No. KR919858, Mayfield et al. unpublished; see also Schmidt-Roach et al. 12 about Pocillopora cf. brevicornis), which differed by 5 bp. A maximum likelihood phylogenetic tree based on mtORF haplotypes was constructed (Fig. 1).
Genetic structure among genetic lineages separated on the basis of mtORF and based on 27 microsatellite loci (Fig. 2), supported the results of Pinzón et al. 10 . Using the method of Evanno et al. 24 to determine the most   probable number of genetic populations (K), the largest peak of ΔK was K = 3 (Fig. 2). Otherwise, ΔK values at K = 5 and K = 8 also showed small peaks; therefore, we showed genetic population structure for values of K ranging from 3 to 8 (Fig. 2). Genetic structure among Type 1 mtORFs was reflected in different ITS2 haplotypes, as reported by Pinzón et al. 10 . We defined ITS2 types T and C within mtORF Type 1, based on their haplotype groups (see registered sequences: Genbank accession No. LC222453-LC222460). Some MLLs in mtORF Type 5 were assigned to the cluster containing Type 4. However, there was only one mtORF Type 4 sample. Further studies with additional samples are needed to fully distinguish these two lineages. Also, when K ≥ 6 in STRUCTURE analysis, definite genetic structure was detected, even between sites within mtORF Type 5, despite the close geographic proximity of these populations, ~17 km along the coast. Thus, genetic structure even in the same genetic lineage at fine geographic scale could be confirmed in a brooding species 25 . In part, genetic differentiation within a given lineage may reflect differences of water depth (<~17 m) between sites.
In conjunction with primers developed previously, these new primer pairs also may be used for a variety of Pocillopora lineages (including various morphological species) and may provide useful information about coral population genetics in general. By sampling a greater number of specimens across a larger geographic area, these microsatellite loci will be beneficial for population genetics within and between Pocillopora lineages in various localities. They will be helpful to understand genetic structure and population dynamics at contemporary and historical time scales, and for parentage analysis, identification of evolutionary lineages, and genetic diversity to understand maintenance of coral populations.
This study suggested that 13 new and 14 previously reported loci are useful as cross-lineage microsatellites for population genetic analyses of Pocillopora in the Ryukyu Archipelago. mtORF Type 5 obtained at two sites for validation of microsatellites indicated large number of clonal replicates, especially in Ueno. We detected distinct genetic structure among lineages, as estimated from mtORF haplotypes (ITS2 was also considered for Type 1). Although Schmidt-Roach et al. 12 reported that at least six lineages of Pocillopora inhabit the northwestern Pacific region, this study indicated that all six occur in the Ryukyu Archipelago, despite sampling only two sites. We confirmed one more lineage with an unknown haplotype in this region. Additional genetic diversity may be discovered in this region by extending the number and geographical distribution of sampling sites. More extensive analyses of genetic diversity and connectivity in within and between populations will be needed to better understand population maintenance and dynamics for conservation and management of coral reef organisms.

Methods
Next-generation sequencing and isolation of microsatellites for two Pocillopora lineages. We collected branches from the morphological species, P. meandrina and P. acuta, along the western coast of Okinawa Island in the Ryukyu Archipelago, a subtropical area located in the northwestern Pacific Ocean, Japan. These belonged to mitochondrial open reading frame (mtORF) Type 1 and Type 5 (see Results and Discussion). Specimens were preserved in ethanol and genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen) following the standard protocol. We sequenced 250-bp paired-end reads using a MiSeq sequencer (Illumina) according to the manufacturer's instructions. After merging single-end sequences using PEAR ver. 0.9.8 26 , to isolate reads of 100 bp or more having repeat sequences of P. meandrina (Type 1), we used MISA (http:// pgrc.ipk-gatersleben.de/misa/) to detect simple sequence repeats and to design PCR primers using Primer3 (http://pgrc.ipk-gatersleben.de/misa/primer3.html). We searched P. acuta (Type 5) using BLAST+ ver. 2.3.0 27 . We confirmed that sequence regions, including microsatellites, were conserved between the two lineages.
Collection of coral samples, sequencing of mtORF, and phylogenetic analysis of Pocillopora. To confirm validation and polymorphism of microsatellite loci, branches from 90 Pocillopora colonies were collected at Ueno (30 colonies; 24°43ʹ09″ N, 125°20ʹ27″ E; <1 m depth, by walking along the seashore) and Yoshino (60 colonies; 24°44ʹ55″ N, 125°26ʹ45″ E; <18 m depth, by SCUBA diving) at Miyako Island at the Ryukyu Archipelago, Japan. Genomic DNA was extracted from preserved coral branches as described above. Mitochondrial haplotypes were confirmed by sequencing the mtORF region 28 . PCR reaction mixtures consisted of: (10 μL) containing <30 ng/μL template DNA, AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific), and the primers (final concentration: 2 μM for each primer) for mtORF: FATP6.1 (5ʹ-TTTGGGSATTCGTTTAGCAG-3ʹ) and RORF (5ʹ-SCCAATATGTTAAACASCATGTCA-3ʹ) 29 . The PCR protocol consisted of 94 °C for 1 min, followed by 40 cycles at 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 75 s, with a final extension at 72 °C for 5 min. After reaction of Exonuclease I (Takara) and Shrimp Alkaline Phosphatase (Takara) to clean up PCR products, sequencing was conducted by Macrogen Japan. mtORF sequences obtained were aligned with others from previous studies 10,13,28,29 including unpublished work of Mayfield et al., using MUSCLE in MEGA ver. 6.06 30 . The molecular evolution model was selected by MEGA, and the best model was determined to be the HKY (Hasegawa-Kishino-Yano) model with a gamma distribution of rate variation across sites (+G). Rapid bootstrap analysis employing the maximum likelihood method for phylogenetic analysis was carried out using MEGA with the model using 1000 bootstrap replicates. The phylogenetic tree was drawn in FigTree ver. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).
Genotyping of Pocillopora using novel and known microsatellite loci. Together with novel microsatellite loci developed here, we characterized loci for Pocillopora previously developed by Magalon et al. 20 , The model criterion of choice to detect the most probable number of genetic clusters (K = 1 to K = 10) across 10 iterations per K (the assumed number of clusters). Graphs of (B) mean log probability (Ln P(D)) and (C) ΔK values based on the rate of change in Ln P (D) between successive K values (K = 2 to K = 9) for detecting the most probable number of K.