Mitogenomic phylogenetic analyses of Leptogorgia virgulata and Leptogorgia hebes (Anthozoa: Octocorallia) from the Gulf of Mexico provides insight on Gorgoniidae divergence between Pacific and Atlantic lineages

Abstract The use of genetics in recent years has brought to light the need to reevaluate the classification of many gorgonian octocorals. This study focuses on two Leptogorgia species—Leptogorgia virgulata and Leptogorgia hebes—from the northwestern Gulf of Mexico (GOM). We target complete mitochondrial genomes and mtMutS sequences, and integrate this data with previous genetic research of gorgonian corals to resolve phylogenetic relationships and estimate divergence times. This study contributes the first complete mitochondrial genomes for L. ptogorgia virgulata and L. hebes. Our resulting phylogenies stress the need to redefine the taxonomy of the genus Leptogorgia in its entirety. The fossil‐calibrated divergence times for Eastern Pacific and Western Atlantic Leptogorgia species based on complete mitochondrial genomes shows that the use of multiple genes results in estimates of more recent speciation events than previous research based on single genes. These more recent divergence times are in agreement with geologic data pertaining to the formation of the Isthmus of Panama.

They reconstructed two phylogenies, one based on complete mitochondrial genomes and the other based on a partial fragment of the mitochondrial MutS gene (mtMutS). While their phylogeny based on complete mitochondrial genomes only has eleven species of the family Gorgoniidae, including six species of Leptogorgia, the one based on the single mtMutS gene includes 109 species, providing greater taxonomic resolution. In their study, Poliseno et al. (2017) also estimate divergence times with a fossil calibration based on the oldest known fossil of Eunicella, dating back to 28.4 Ma (Kocurko & Kocurko, 1992) using the partial mtMutS gene. Based on their results, Poliseno et al. (2017) call for a global taxonomic revision of the present-day Leptogorgia genus. They conclude from the mtMutS phylogeny that the genus Lophogorgia should be resurrected for all South African Leptogorgia species, which form an old clade within the Gorgoniidae, sister to Leptogorgia species from the eastern coast of Africa and the Mediterranean. They show that these Eastern Atlantic Leptogorgia species diverged from Western Atlantic species in the late Cretaceous, about 65 Ma, while the divergence between the Western Atlantic and Eastern Pacific species occurs more recently, between 28 and 23 Ma. These observations not only raise the question of taxonomic placement and nomenclature for Eastern Pacific and Western Atlantic species, but also suggest a divergence time between these lineages that dates back to the very early stages of emergence of the Isthmus of Panama (Bacon et al., 2015). This scenario is unexpected since Leptogorgia are shallow water species and significant exchange of seawater between the two basins likely occurred until ~10-15 Ma when the final stages of the closure of the Central American Seaway (CAS) started, with shallow water still connecting these two oceans until 3.5-4.2 Ma with the final rise of the Isthmus of Panama (e.g., Bacon et al., 2015;Montes et al., 2015;O'Dea et al., 2016).
Our study focuses on two species of Leptogorgia from the Gulf of Mexico, L. hebes and L. virgulata. We have two main goals. The first is to determine the taxonomic position of L. hebes (formerly classified in the genus Lophogorgia by Bayer, 1961) and of L. virgulata. The analyses by Poliseno et al. (2017) did not include complete mitochondrial genomes for these two species and their phylogeny based on the partial mtMutS gene leaves the phylogenetic position of both L. hebes and L. virgulata weakly supported. Therefore, in our study, we analyze both complete mitochondrial genomes and the mtMutS gene.
Our second goal is to estimate divergence times of Eastern Pacific and Western Atlantic Leptogorgia species. Since previous research has shown that fossil-calibrated phylogenetic reconstruction based on single mitochondrial genes results in an overestimation of divergence times (Duchêne, Archer, Vilstrup, Caballero, & Morin, 2011;McCormack, Heled, Delaney, Peterson, & Knowles, 2011), we will base our estimates of diversification times between Eastern Pacific and Western Atlantic lineages of Leptogorgia by targeting complete mitochondrial genomes. We reconstruct a fossil-calibrated phylogenetic tree for Leptogorgia species based con complete mitochondrial genomes and using Eunicella as an outgroup. We use a fossil calibration point of 28.4 Ma based on the stratigraphy and dating of the Red Bluff Formation in Mississippi where the oldest fossils of Eunicella have been recovered (Cushing, Boswell, & Hosman, 1964;Demchuk & Gary, 2009;Kocurko & Kocurko, 1992;Prothero, Ivany, & Nesbitt, 2003;Tew, 1992). Among Octocorallia, skeletal diversity, such as morphology of sclerites, is a key character for taxonomic identification (Goffredo & Dubinsky, 2016). Sclerites with a balloon club shape are a distinguishing characteristic that is unique to the genus Eunicella (Goffredo & Dubinsky, 2016;Kocurko & Kocurko, 1992). Fossil sclerites with balloon club shape have been found in the Red Bluff Formation in Mississippi and have been clearly attributed to Eunicella (Kocurko & Kocurko, 1992). Stratigraphy of the Red Bluff Formation and dating of this layer within the Oligocene (23-34 Ma) has been intensely studied (i.e., Cushing et al., 1964;Demchuk & Gary, 2009;Hosman, 1996;Prothero et al., 2003;Tew, 1992).
The timeline proposed by Poliseno et al. (2017) for the divergence between Eastern Pacific and Western Atlantic Leptogorgia species coincides with evidence that a land bridge between North and South America began to emerge between 23 and 25 Ma when the Panama Arc collided with South America (Bacon et al., 2015). However, despite this initial emergence and given the life history characteristics of shallow water Leptogorgia species such as L. hebes and L. virgulata that enhance dispersal and colonization (Beasley & Dardeau, 2003;Cairns & Bayer, 2009;Gotelli, 1988Gotelli, , 1991Williamson et al., 2011), gene flow is likely to have continued between the Western Atlantic and Eastern Pacific until full closure of the Central American Seaway (Bacon et al., 2015;Cowman & Bellwood, 2013;Lessios, 2008;Thacker, 2017).. Therefore, we hypothesize that the divergence times of Eastern Pacific and Western Atlantic Leptogorgia lineages to be younger than previously suggested (Poliseno et al., 2017) with the majority of speciation events occurring after 10 Ma when significant seawater exchange between the Pacific and Atlantic Ocean ceased (i.e., Bacon et al., 2015;Montes et al., 2015;O'Dea et al., 2016).

| DNA Extraction and PCR
Three to five individual polyps were picked off from each coral sample, depending on the size and quality of preservation of the coral fragment. Polyps were visually inspected under a stereo microscope and picked off the coral stalk using forceps. Forceps were sterilized in between each sample using 100% bleach and 100% ethanol. If individual polyps were difficult to distinguish, an ~0.5 cm long piece was broken off of the coral fragment. The PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific) was used to extract DNA from each sample following the manufacturer's standard protocol. Prior to extraction, coral polyps were rehydrated for 1-2 hr in molecular grade water and then digested for at least 5 hr. The final DNA product was eluted two times for maximum yield. The elution buffer was heated to 55°C prior to use, and 60 µl of were used for both elutions. The concentration of the extracted DNA was measured using a Qubit fluorometer (Life Technologies Inc.
Eugorgia mutabilis c for 1 min, 50°C for 1 min, 72°C for 1 min, and a final step at 72°C for 5 min. The resulting product was visualized by gel electrophoresis on an ultraviolet light transilluminator to assess DNA length and quality. Once all samples yielded successful amplification, the final PCR product was then purified with the Invitrogen PureLink PCR Purification Kit, following the manufacturer's procedure. The primers and purified PCR products were then sent to Eurofins Genomics for sequencing of forward and reverse strands.
The genomic DNA extraction from each specimen was visualized after gel electrophoresis with an ultraviolet light transilluminator.
Genomic DNA of high molecular weight with minimal degradation was identified by looking for high concentrations above 5,000 bp, with minimal streaking below this size. Based on these observations, the mitochondrial genome of ten specimens with the highest quality of genomic DNA was targeted using next generation sequencing technology. The genomic DNA extraction of these ten specimens was used to prepare an indexed library following standard proce-

| Sequence assembly and alignment
For each specimen, the sequences for the forward and reverse strands were assembled with the software CLC Workbench 7.9.1 (CLC Bio) using the settings: minimum aligned read length = 500 bp, alignment stringency = high, conflicts = ambiguity nucleotides, trim sequence ends and trim using quality scores limit = 0.05. A cutoff was used were only bases with Phred scores of 20 or more were kept. A consensus sequence was generated from each assembly.
Qiagen's CLC Workbench 7.9.1 was used to align the mtMutS sequences. The mtMutS sequences were aligned using Qiagen's CLC Main Workbench 7 software and include 24 sequences from this study, the 114 sequences examined in Poliseno et al. (2017) and 43 novel sequences available in GenBank for a total of 182 sequences ( Table 2). The alignment was visually inspected for errors and inconsistencies. The final mtMutS alignment was 766 bp in length.
The Illumina sequence reads were assembled using the software CLC Genomics Workbench 11. Default settings were used with reads mapped back to contigs (mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.5, similarity fraction = 0.8).
The sequences obtained from the assemblies included the full mi- Sequences from this study.
b New sequences from GenBank. c Sequences used by Poliseno et al. (2017).

TA B L E 2 (Continued)
genomes were annotated using Qiagen CLC Genomics Workbench 11 software using previously published Leptogorgia mt genomes as references (Table 3). The ten mitochondrial genomes obtained were analyzed along with eleven mitochondrial genomes available in GenBank (Table 3). Individual genes and RNAs were extracted and aligned separately using MUSCLE v3.8 (Edgar, 2004)

| Phylogenetic analyses
Both mtMutS and complete mt genome alignments were used in phylogenetic analyses using maximum likelihood (ML) and Bayesian methods. The model of evolution and partitioning scheme was determined by PartitionFinder v1.1.1 (Lanfear, Calcott, Kainer, Mayer, & Stamatakis, 2014) using linked branches and the Akaike information criterion (AIC). The RAxML v8.0.0 program (Stamatakis, 2017) was used to conduct the ML analyses and Mr. Bayes 3.1 (Ronquist & Huelsenbeck, 2003) was used for the Bayesian analyses. Data blocks were created for mtMutS based on codon position (Table 4).
Divergence time estimates were performed by Bayesian analyses using full mitochondrial genomes only, with the software BEAST 2.3.2 (Bouckaert et al., 2019). The alignment was partitioned as specified above for the Bayesian phylogenetic reconstruction (Table 3).
An uncorrelated log-normal relaxed clock model was used along with the calibrated yule speciation model. The tree was calibrated based on the earliest fossil evidence for Eunicella (Kocurko & Kocurko, 1992) with a date of origination set to 28.4 Ma (mean = 1 and standard deviation = 1). One chain was carried out for 10,000,000 generations, sampling every 1,000th generation. After inspecting the trace files generated by the Bayesian Markov Chain Monte Carlo (MCMC) runs, the initial 25% of sampled generations were omitted prior to building the tree. Mean divergence times were summarized with TreeAnnotator.

| Mitogenomic phylogeny
The phylogenetic reconstruction based on full mitochondrial genomes included 7 mt genomes of L. virgulata and 3 mt genomes of L. hebes generated by this study. These mt genomes were combined with 11 additional mt genomes from the family Gorgoniidae and two mt genomes of Eunicella (outgroup), downloaded from GenBank (Table 3)

| Mitogenomic divergence time estimation
The phylogenetic reconstruction based on mitochondrial genomes using fossil-calibrated coalescent methods as implemented by

| Mitochondrial MutS phylogeny
The reconstructed mtMutS phylogeny uses 68 new mtMutS sequences (24 from this study and 44 from GenBank) added to the sequences used in the phylogenetic tree by Poliseno et al. (2017).
This new mtMutS phylogeny agrees with the phylogeny presented by Poliseno et al. (2017). The Leptogorgia species from South Africa form a sister clade to species from the Eastern Atlantic and Mediterranean while the latter three are Western Atlantic species. Further morphological and genetic analyses of these species in particular will be necessary in order to more accurately classify them and determine whether resurrecting the genus Lophogorgia would be appropriate.

| Mitogenomic phylogeny
The complete mitochondrial genomes of 21 gorgonian specimens were examined to elucidate phylogenetic relationships and to test the efficacy of using complete mt genome over the single mtMutS gene. This is the first study to sequence complete mitochondrial genomes for L. virgulata and L. hebes, and the resulting mitogenomic phylogeny is in agreement with our mtMutS phylogeny and with that of Poliseno et al.'s (2017), albeit with stronger branch support. The tree topology also matches that of the mitogenomic phylogeny presented by Poliseno et al. (2017)

| Divergence time estimation
This is the first study to place divergence time estimates on complete mitochondrial genomes of Leptogorgia species. Poliseno et al. Pacific oceans (O'Dea et al., 2016). Both, L. hebes and L. virgulata are adapted to shallow water habitat ranging from 3 to 82 m (Cairns & Bayer, 2009;Williamson et al., 2011). They mature rapidly (<2 years) and are broadcasts spawners, releasing eggs and sperm into the water column (Beasley & Dardeau, 2003;Gotelli, 1991). While larval duration in L. hebes is not known, it can last up to 20 days in L. virgulata (Gotelli, 1991). These characteristics indicate a potential for high dispersal and suggest that gametic and larval connectivity The divergence times obtained from this study are more recent than those presented by Poliseno et al. (2017) and with lower error estimates (2-4 million-year range as opposed to a 12-40 million-year range). This discrepancy is most likely attributed to our use of complete mitochondrial genomes that include fourteen protein-coding genes and two RNAs instead of a single, partial gene (mtMutS). There are numerous studies of multiple taxa showing a pattern of incongruent tree topology between single mitochondrial markers and complete mitochondrial genomes despite the fact that they are the same locus and therefore share the same phylogenetic history Knaus et al., 2011;Luo et al., 2011;Nadimi et al., 2016;Pacheco et al., 2011;Rohland et al., 2007;Urantowka et al., 2017;Wang et al., 2017;Willerslev et al., 2009). For example, Havird, Santos Scott, and Schierwater, (2014) analyze the performance of single and concatenated sets of mitochondrial genes relative to complete mitochondrial genomes for phylogenetic reconstruction of metazoans. Their findings show that single genes are not able to reproduce the topology of a mitogenomic phylogeny . A similar study, but focusing on birds, showed that single mitochondrial genes resulted in incorrect and contradictory phylogenetic relationships, while the use of complete mitochondrial genomes accurately reflected the species tree (Urantowka et al., 2017). The same pattern has been observed in insects, where individual mitochondrial genes can result in different and contradicting tree topologies, while using the complete mitochondrial genome performs well at various taxonomic levels (Wang et al., 2017). In fungi, the phylogenetic signal differs between single mitochondrial genes, subsets of concatenated mitochondrial genes, and complete mitochondrial genomes, despite all being the same locus (Nadimi et al., 2016). it is difficult to distinguish between species when using the single gene mtMutS and that even using a concatenated set of 2-3 different mitochondrial regions only allows to distinguish 70%-80% of morphological species (i.e., Baco & Cairns, 2012;McFadden et al., 2011). The low resolution provided by the use of a single mitochondrial region explains the low support for many clades in the mtMutS phylogeny presented in this study and that of Poliseno et al. (2017). Greater resolution and strong support of clades within the Octocorallia is achieved by using complete mitochondrial genomes, as demonstrated in our present study and in previous research (i.e., Figueroa & Baco, 2015Kayal et al., 2013;Poliseno et al., 2017).
In addition to incongruent topologies and weakly supported clades, the use of single genes can result in overestimation of calibrated divergence times (Duchêne et al., 2011;McCormack et al., 2011 (Duchêne et al., 2011). These results from previous research are congruent with our observations that in octocorals, such as the gorgonians analyzed in our study, the use of complete mitochondrial genomes as opposed to single mitochondrial genes, results in better resolved, well supported, trees that have earlier and more precise divergence time estimates. Since our divergence time estimates are concordant with regional geological events and divergence patterns of other organisms, it supports our hypothesis that the divergence times of Eastern Pacific and Western Atlantic Leptogorgia lineages is younger than previously suggested (Poliseno et al., 2017) with the majority of speciation events occurring after 10 Ma when significant seawater exchange between the Pacific and Atlantic Ocean ceased (e.g., Bacon et al., 2015;Montes et al., 2015;O'Dea et al., 2016). However, future work that includes multiple nuclear markers in addition to mitochondrial genomes is necessary to fully test this hypothesis.

O PE N R E S E A RCH BA D G E S
This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https ://www.ncbi.nlm.nih.

DATA AVA I L A B I L I T Y S TAT E M E N T
Mitochondrial genome and mtMutS sequences can be accessed online through GenBank (accession numbers listed in Table 4).