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Article

Phylogenomic Insights into the Phylogeography of Halophila baillonii Asch.

by
Kor-jent van Dijk
1,2,
Michelle Waycott
1,2,
Ed Biffin
2,
Joel C. Creed
3,
Federico J. Albertazzi
4,5 and
Jimena Samper-Villarreal
6,*
1
School of Biological Sciences, Department of Ecology and Evolutionary Biology and the Environment Institute, University of Adelaide, Adelaide 5005, Australia
2
State Herbarium of South Australia, Botanic Gardens and State Herbarium, Department for Water and Environment, Adelaide 5005, Australia
3
Instituto de Biologia Roberto Alcântara Gomes Rio de Janeiro, Universidade do Estado do Rio de Janeiro, Maracanã 20550-900, Brazil
4
Centro de Investigación en Biología Celular y Molecular (CIBCM), Ciudad de la Investigación, Universidad de Costa Rica, San José 11501-2060, Costa Rica
5
Escuela de Biología, Sede Rodrigo Facio, Universidad de Costa Rica, San José 11501-2060, Costa Rica
6
Centro de Investigación en Ciencias del Mar y Limnología (CIMAR), Ciudad de la Investigación, Universidad de Costa Rica, San José 11501-2060, Costa Rica
*
Author to whom correspondence should be addressed.
Diversity 2023, 15(1), 111; https://doi.org/10.3390/d15010111
Submission received: 9 November 2022 / Revised: 16 December 2022 / Accepted: 20 December 2022 / Published: 13 January 2023
(This article belongs to the Special Issue Ecology, Diversity and Conservation of Seagrass)
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Abstract

:
A molecular genetic approach was used to elucidate the phylogeographic relationships of the clover grass (Halophila baillonii Asch.) from three key regions within its current distributional range. Halophila baillonii is a small seagrass that has historically been only found in a few locations in the Caribbean and Atlantic coast of Brazil. In the past few decades H. baillonii has also been observed in the Eastern Tropical Pacific (ETP). Very little is known about the relationship between the ETP populations and the Caribbean and Atlantic ones. To study their relationship, we used a hybrid capture approach targeting chloroplast loci on samples from Belize, Brazil, and the Pacific coast of Costa Rica. Phylogenetic analyses resolved H. baillonii as monophyletic and placed the samples from Belize and Costa Rica in a clade sister to the Brazilian ones. The results clearly indicate that the ETP samples are very closely related to Belize and that the most likely explanation of its occurrence in the ETP is a recent introduction, possibly as a consequence of the opening of the Panama Canal.

1. Introduction

Seagrass meadows, including those dominated by smaller seagrass species such as Halophila are a key coastal habitat and provide many ecosystem services. For example, these small seagrasses serve as habitat for fish and invertebrates, provide sediment stabilization, water purification and serve as a food source for megaherbivores such as dugongs [1]. Halophila species are considered to be colonizing seagrasses [2,3], where despite low resistance to perturbations they have substantial capacity to grow back following loss. This recovery potential is linked to having fast shoot turnover rates, a short sexual maturity period, and production of a large number of seeds able to persist in a dormant state thereby forming a seed bank in the sediment [3]. Their capacity for recovery means that Halophila species play a key role in disturbed or highly dynamic seagrass meadows often acting as the ruderal occupants in these sites [3].
Halophila species are found in most seagrass bioregions around the world, being absent only in the Temperate North Pacific and Temperate North Atlantic regions [4]. Some Halophila species, such as Halophila decipiens Ostenf., have a very wide distribution, being found in all four bioregions where Halophila is present [4]. Other species are only found in the southern temperate waters of Australia, such as Halophila australis Doty & B.C. Stone [5]. Species of Halophila are easy to recognize as the only seagrass genus with ovate and usually petiolate leaves. Leaf shape is one of the main morphological characteristics that is used for species identification. However, the leaves of some Halophila species look very similar to each other and other characters such as leaf venation, leaf margin, and leaf hairs have been used to discriminate species. The Halophila ovalis (R.Br.) Hook.f. complex is particularly controversial as key characters (such as leaf size and shape) overlap and do not match the phylogeny e.g., [6]. On the other hand, some Halophila species are easier to recognize as these have higher leaf numbers clustered at each node, such as Halophila spinulosa (R.Br.) Asch., Halophila tricostata M.Greenway, Halophila engelmanii Asch., and Halophila baillonii Asch.
The focus of this study is H. baillonii, commonly known as “clover grass”. In some instances, the species is likely to have been reported as Halophila baillonis, previously considered a synonym for H. baillonii [7]. This species has a distribution that is largely confined to the Tropical Atlantic seagrass bioregion [4,7]. It is easily recognizable with vertical stems arising from the rhizome that terminate in two pairs of serrated oval shaped leaves. The species is dioecious and flowers form in the center of the leaf whorls [8].
Halophila baillonii has been assessed as having a decreasing population trend and is currently categorized as Vulnerable under IUCN Red List criteria [9]. While largely restricted to the Caribbean and the Atlantic coast of Brazil, it has unexpectedly been reported in several locations in the Eastern Tropical Pacific (ETP) [7,10] (Figure 1). Seagrasses are rarely reported to occur along the tropical Pacific coastline of central America south of Baja California, Mexico. The first sightings of H. baillonii in the ETP were in Panama in the 1970s [11]. Further sightings of this species in the ETP were reported in the mid 1990s on the Pacific coast of Costa Rica, at a meadow that vanished following a storm event shortly after its discovery [11]. In recent years, however, H. baillonii has been reliably found along the Pacific coast of Costa Rica [10,12,13,14,15,16] and has now also been reported in Nicaragua [17]. Given that the first report of this species in the Pacific was in the 1970s, a reasonable inference is that this is a recent introduction, most likely from the Caribbean to the ETP, but this idea has yet to be investigated.
It would be reasonable, however, to hypothesize that there is more than one alternative scenario to consider. The first is a remnant species distribution hypothesis, where the current restricted and fragmented distribution of H. baillonii are remnants of an historically more widespread range. Given this, we would expect the populations in different regions to differ genetically from each other due to isolation over time. Under this model, there would be an assumption that ETP populations established before the formation of the Isthmus of Panama around 2 to 3 Ma and it would be inferred that the species was widespread across areas that were connected to both coasts of what is now Central America [18]. The creation of a land barrier would have limited geneflow between both water bodies eventually due to the total closure of the Isthmus. It is expected that 2–3 M years of isolation would lead to significant divergence and result in a strong phylogenetic signal between the H. baillonii in the Caribbean and Atlantic compared to the ETP. We would also expect the Atlantic populations to have a higher affinity with the Caribbean ones, as gene flow between these two is historically likely to occur. It is also likely that extant populations have very low diversity remaining where dispersal–recruitment events or gradients have occurred leading to random genetic drift and further genetic divergence.
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Figure 1. Locations where Halophila baillonii populations have been reported in the Caribbean and Atlantic (Green) [19] and recently in the Eastern Tropical Pacific (Maroon). Sites collected for this study are shown as black dots: Costa Rica (5 sites), Brazil (2 sites), and Belize (1 site). See Table 1 and Supplemental Table S1 for further details on samples from each site.
Figure 1. Locations where Halophila baillonii populations have been reported in the Caribbean and Atlantic (Green) [19] and recently in the Eastern Tropical Pacific (Maroon). Sites collected for this study are shown as black dots: Costa Rica (5 sites), Brazil (2 sites), and Belize (1 site). See Table 1 and Supplemental Table S1 for further details on samples from each site.
Diversity 15 00111 g001
Table
Table 1. Name and origin of Halophila samples used in phylogeny. Additional information can be found in Supplemental S1. * Provided for H. baillonii only.
Table 1. Name and origin of Halophila samples used in phylogeny. Additional information can be found in Supplemental S1. * Provided for H. baillonii only.
#Sample Name SpeciesCountry of OriginCoastline *Sampling Site
1H. baillonii Belize 1H. bailloniiBelizeCaribbeanPlacencia
2H. baillonii Belize 2H. bailloniiBelizeCaribbeanPlacencia
3H. baillonii Belize 3H. bailloniiBelizeCaribbeanPlacencia
4H. baillonii Belize 4H. bailloniiBelizeCaribbeanPlacencia
5H. baillonii Belize 5H. bailloniiBelizeCaribbeanPlacencia
6H. baillonii Belize 6H. bailloniiBelizeCaribbeanPlacencia
7H. baillonii Belize 7H. bailloniiBelizeCaribbeanPlacencia
8H. baillonii Costa Rica 1.1H. bailloniiCosta RicaPacificGolfo Dulce, Refugio Animal
9H. baillonii Costa Rica 2.1H. bailloniiCosta RicaPacificGolfo Dulce, Playa Colibrí
10H. baillonii Costa Rica 2.2H. bailloniiCosta RicaPacificGolfo Dulce, Playa Colibrí
11H. baillonii Costa Rica 2.3H. bailloniiCosta RicaPacificGolfo Dulce, Playa Colibrí
12H. baillonii Costa Rica 2.4H. bailloniiCosta RicaPacificGolfo Dulce, Playa Colibrí
13H. baillonii Costa Rica 3.1H. bailloniiCosta RicaPacificBahía Matapalito
14H. baillonii Costa Rica 3.2H. bailloniiCosta RicaPacificBahía Matapalito
15H. baillonii Costa Rica 3.3H. bailloniiCosta RicaPacificBahía Matapalito
16H. baillonii Costa Rica 4.1H. bailloniiCosta RicaPacificBahía Potrero
17H. baillonii Costa Rica 4.2H. bailloniiCosta RicaPacificBahía Potrero
18H. baillonii Costa Rica 5.1H. bailloniiCosta RicaPacificSámara
19H. baillonii Costa Rica 5.2H. bailloniiCosta RicaPacificSámara
20H. australis Australia 1H. australisAustralia-Port Noarlunga
21H. australis Australia 2H. australisAustralia-SA Pearsons Sanctuary Zone
22H. australis Australia 3H. australisAustralia-VIC Flinders Island
23H. beccari SingaporeH. beccariSingapore-Mandi
24H. decipiens Australia H. decipiensAustralia-QLD Lowe Isles
25H. decipiens SpainH. decipiensSpain-Canary Islands El Hierro
26H. engelmanii MexicoH. engelmaniiMexico-Puerto Progreso
27H. engelmanii USAH. engelmaniiUSA-Florida Black Water Sound
28H. ovalis Australia 1H. ovalisAustralia-WA Rottnest Island Parker Bay
29H. ovalis JapanH. ovalisJapan-Okinawa
30H. ovalis AntiguaH. ovalisAntigua and Barbuda-Antigua
31H. ovalis Australia 2H. ovalisAustralia-WA Two People Bay
32H. ovalis MadagascarH. ovalisMadagascar-Andrevo Tulear
33H. ovalis MauritiusH. ovalisMauritius-Flic en Flac
34H. spinulosa SingaporeH. spinulosaSingapore-Check Java
35H. stipulacea ZanzibarH. stipulaceaTanzania, Zanzibar-Chwaka Bay
36H. stipulacea Virgin IslandsH. stipulaceaUSA, Virgin Islands-St Thomas
37H. stipulacea UAEH. stipulaceaUnited Arab Emirates-Mubarraz Island
38H. tricostata AustraliaH. tricostataAustralia-QLD Lowe Isles
39H. baillonii Brazil 1.1H. bailloniiBrazilAtlanticPonta do Socó, Praia das Pedras
40H. baillonii Brazil 1.2H. bailloniiBrazilAtlanticPonta do Socó, Praia das Pedras
41H. baillonii Brazil 1.3H. bailloniiBrazilAtlanticPonta do Socó, Praia das Pedras
43H. baillonii Brazil 2.1H. bailloniiBrazilAtlanticBarra do Mamanguape, Banco d’areia
44H. baillonii Brazil 2.2H. bailloniiBrazilAtlanticBarra do Mamanguape, Banco d’areia
45H. baillonii Brazil 2.3H. bailloniiBrazilAtlanticBarra do Mamanguape, Banco d’areia
The alternative hypothesis is that H. baillonii is a recent introduction into the ETP. There are multiple pathways and vectors through which this could have occurred. One possibility is the drifting/rafting of plant fragments from one ocean to the other via the Panama Canal, which was opened in 1914. However, the canal has locks and the inner sections are composed of freshwater so this seems unlikely. A second alternative is that the opening of the Panama Canal increased ship traffic, and that plant fragments or seeds were moved to the Pacific by large ships (e.g., on anchors, solid ballast, or ballast water). Seeds or plants could also be carried on small watercraft (e.g., on anchors or in storage lockers) hauled between seaboards. Recent biotic dispersal is also possible, and for example birds could have moved seeds from the Caribbean to the ETP, covering a distance of only ~80 km. This seems to be unlikely, however, as it could have occurred many times during the last 2–3 M years, and we would then expect that H. baillonii and other Caribbean seagrass species would be more common in the ETP. Under the premise that H. baillonii is a recent introduction to the ETP, we expect low genetic divergence between these populations and the source population(s) in the Caribbean and/or Atlantic. We would also expect the Caribbean and Atlantic populations to have higher genetic differentiation than the ETP.
This study aimed to assess whether H. baillonii populations in the ETP and those in the Caribbean and Atlantic are conspecific. Furthermore, we attempt to identify the origin of the disjunction in H. baillonii across the Isthmus of Panama. To further these aims, we use a phylogeographic genomic approach where multiple chloroplast gene regions were sequenced using hybridization capture.

2. Materials and Methods

2.1. Sampling, DNA Extractions and Library Preparation

Phylogeographic analyses were conducted on a total of 25 H. baillonii samples that were collected from Costa Rica in the ETP (12 samples), Brazil in the Atlantic (6 samples) and Belize in the Caribbean (7 samples, see Table 1 and Figure 1). Additionally, plant material from seven other Halophila species were added to the sample set to assess phylogenetic vs. phylogeographic distances between species and populations (Table 1). Samples of H. baillonii from Costa Rica and Brazil were specifically collected for this study. We have made repeated attempts over the past 10 years to obtain samples of H. baillonii from other locations within the current distribution range of this species. However, our endeavors have proven extremely challenging given the limited abundance of this species in the field and of known current localities for active sampling. The remainder of the Halophila samples are part of a large dry sample collection that has been gathered over the past 25 years in the Waycott lab in Adelaide, Australia. Brazilian samples were collected under licence No. 80652-1 and genetic resources exported under SisGen/CGEN/MMA number R9C7FD8.
DNA extractions from Belize and Brazil were performed using the DNeasy® Plant Mini Kit (Qiagen) starting with ~10 mg of tissue. Grinding was done on a Bead Ruptor 24 (OMNI International Inc.) in 2 mL screw cap tubes. Initial lysis steps were done manually and after the ice precipitation step extractions were finalized on a QIAcube HT (Qiagen) following the manufacturer’s instructions. Final DNA elutions were done in 100 µL EB buffer. Samples from Costa Rica were extracted locally using NucleoSpin Plant II (Macherey-Nagel), ground with liquid nitrogen starting with ~100 mg of dry tissue according to manufacturer’s instructions. Total DNA was eluted two times with 25 µL PE buffer. The concentration and purity of DNA extracts were measured using a spectrophotometer (NanoDrop 2000c) and samples were sent to Adelaide, Australia for analysis.
For phylogenetic and phylogeographic data sets the myBaits® target capture kits (Daicel Arbor Biosciences, Ann Arbor, MI, USA) were used to isolate target genes. Illumina sequencing libraries and RNA bait captures were performed using the method described in Waycott, et al. [20]. For this study, a refined version of OzBaits chloroplast bait (myBaits design ID: D10125PtCOI) set was used to isolate informative loci [20], consisting of ~50 chloroplast loci instead of 19 loci (Design ID: D10122Plstd). Library preparation was done with the NEBNext® Ultra™ II FS DNA Library Prep kit with Fragmentase and Sample Purification Beads (New England Biolabs, Ipswich, MA, USA). Neat DNA extracts were used as the starting material and reactions were done in 1/3 volumes see details in [20]. To enable bioinformatics processing following myBaits capture, Y-adaptors (compatible to Illumina TruSeq primers) with synthetic “barcodes” were annealed to the ends of the DNA fragments. These barcodes allow for samples to be identified by a unique synthetic 8 nt DNA barcode situated on the 3′ end of the Read 1 and 2 primer binding sites. Hybrid capture was performed following myBaits manufactures manual (chemistry v5.0), hybridization was done at 63 °C and incubated for 24 h. Post capture PCR was performed on the half build libraries to fuse the remaining sequencing adapters to the ends of the DNA fragments [20], as this library was sequenced with other libraries on the same Illumina lane 8nt unique P5 and P7 indexes were inserted. Libraries were pooled in equimolar concentrations and size selected to 350–600 bp with a Pippin Prep 2% agarose cassette (Sage Science, Beverly, MA, USA). Subsequently they were quantified on a 2100 Bioanalyzer (Agilent) using the high sensitivity DNA assay and molarity was calculated between 300 and 800 bp. Final libraries were sent for Illumina paired-end sequencing (2 × 150) on a NovaSeq 6000 S1 lane to the Australian Genome Research Facility (AGRF) in Melbourne.

2.2. Post Sequencing Data Processing Targeted Genes

High-throughput 150 bp paired-end reads were processed using CLC Genomics Workbench v12 (https://www.qiagenbioinformatics.com, accessed on 27 May 2022). Reads were demultiplexed and named based on the 8 nt barcodes followed by trimming to remove residual adapter sequences and low-quality base calls (Phred-score threshold of 20). For the chloroplast genome recovery, a mapping reference was generated by mapping the reads of a single H. baillonii sample (GIBS 1973) to the chloroplast genome of Halophila beccari Asch. (NCBI accession number MN843749; CLC conditions 85% of length of each read had to map with 95% similarity). Consensus sequences were extracted using a coverage cut-off of 20, consensus length of ≥400 nt and using ‘vote’ to resolve conflicts (a total of 33 regions were retained, Supplemental S2). Reads from each sample were then mapped against this reference (75% of length of each read had to map with 90% similarity) and consensus sequences extracted with a read threshold of 10 and calling ambiguous bases (Ns) below this cut-off. Consensus sequences for each sample were imported into Geneious Prime v2022 [21]. Alignments were generated for the 33 chloroplast regions using the MUSCLE [22] plugin for Geneious with default parameters and 24 regions were kept for further analyses. The concatenated alignment of the 24 chloroplast regions (Supplemental S3) was analyzed using the maximum likelihood phylogenetic software IQ-TREE 2 [23] with the best model estimated by ModelFinder [24] and branch support assessed with 1000 ultra-fast bootstrap (UFB) replicates [25]. A haplotype network was generated using a H. baillonii only sample set (see Table S1 and alignment; Supplemental S4) in PopArt (http://popart.otago.ac.nz, accessed on 4 June 2022) using the Median-joining algorithm [26] for network inference. For these analyses we first masked all ambiguous sites in the concatenated alignment to reduce the impacts of missing values.

3. Results

Sequencing outputs for the sample libraries varied between 200,000 to 4 M reads. Trimmed unprocessed reads can be found in the SRA database from NCBI (Accession numbers available in Table S1). Phylogenetic analyses of the chloroplast data resolved H. baillonii as monophyletic with strong statistical support (UFB ≥ 95%; [27]) and placed the samples from Belize (Caribbean) and Costa Rica (Pacific) in a clade that is sister to the Brazilian (Atlantic) samples (Figure 2 and Supplemental S5 for H. engelmanii and H. baillonii only). Network analyses of the masked alignment indicates that of 25 segregating sites 21 separate the Brazilian from the Caribbean and Costa Rican samples (Supplemental S6).

4. Discussion

This study investigated the phylogeographic relationships of Halophila baillonii from three key regions representing the extent of the current distributional range for this seagrass species. We aimed to establish if the occurrences of H. baillonii on the Pacific coast of Costa Rica in the Eastern Tropical Pacific (ETP) are: (1) remnant populations that established before the closing of the Panama Isthmus; or (2) they are the result of more recent dispersal and establishment from the Tropical Atlantic and/or Caribbean. The phylogeographic analysis demonstrated that H. baillonii from the Caribbean, Atlantic, and the ETP are genetically closely related and group separately from the other Halophila species analyzed (Figure 2 and Supplemental S6). If we accept a vicariance hypothesis, the divergence across the Panama Isthmus would be in the order of 3 M years (i.e., the timing of the seaway closure [18]). In light of recent molecular clock estimates for Halophila e.g., [28], this period of separation is similar to those estimated for the divergence among sister species and among major infra-specific lineages within the genus. If we are willing to accept these estimates, the limited genetic differentiation between the H. baillonii in the Caribbean, Atlantic, and in the ETP favors a hypothesis that populations from these regions separated substantially more recently than the formation of the Panama Isthmus.
The H. baillonii samples from the Atlantic are approximately 5500 and 7000 km distant from the Caribbean ones (Figure 1) and show a much higher level of genetic divergence relative to differences between the Caribbean and ETP ones (Figure 2 and Supplemental S6). While the origin of this divergence is unclear, we suggest that accepting the first hypothesis, above, would suggest an implausibly ancient separation of the Atlantic from the Caribbean lineages. One potential driver for the observed level of differentiation is the Amazon–Orinoco Plume, a major biogeographic barrier between the Caribbean and the Brazilian sites south of the Amazon sampled in the Atlantic, corresponding to the freshwater and sediment outflows of these major river systems e.g., [29]. This plume has been shown to be an important barrier to animal dispersal, particularly through the Pleistocene (c. 2.5–0.01 M years ago) e.g., [30], although its effect on seagrass dispersal is unclear. It remains possible that the distribution range of H. baillonii was more extensive in the past and that the Atlantic populations have become isolated over time.
Many Halophila species appear to be capable of very long-distance dispersal given their very widespread distribution. For example, H. decipiens is found globally and has very low genetic diversity among bioregions, implying regular and long-distance dispersal [6,31]. Other Halophila species have quickly and successfully colonized previous unoccupied coastal areas or seas. Halophila ovalis for example, which is not known to occur naturally in the Atlantic, has ‘recently’ been translocated to the Eastern coast of Florida. A comprehensive study by Waycott et al. [6] demonstrated that Halophila johnsonii Eiseman is conspecific with H. ovalis and is most likely an introduction from an east African population. This study also found that the Floridian population might be one huge clone. Another example is Halophila stipulacea (Forsskål and Niebuhr) Asch., a species that was originally only known to occur in the Indian Ocean, the Persian Gulf and the Red Sea [19]. However, H. stipulacea invaded the Mediterranean Sea about 150 years ago following the opening of the Suez Canal. In 2002, the species was reported for the first time in the Caribbean islands, quickly reaching the South American mainland [32,33,34]. It is therefore quite conceivable that H. baillonii has crossed to the Pacific Ocean and has established.
The above-mentioned ‘recent’ range expansions are most likely due to human mediated mechanisms as opposed to the natural dispersal pathways of these plants. These include the opening of shipping canals (e.g., Panama Canal and Suez Canal) connecting previously discrete waterbodies, and the movement of biological material by ships, increasing the dispersal potential beyond the species natural limits. We believe that the most likely origin for H. baillonii dispersal to Costa Rica (as a Phase II dispersal event [35]) is via ship anchor, solid ballast or ballast water through the Panama Canal, as this is the fastest and potentially less physiologically stressful mode of dispersal. We cannot immediately discount the possibility that fragments of the seagrass were dispersed by superficial currents through the Panama Canal, but the internal waters of Gatun Lake are fresh, making survival far more challenging and thus unlikely. Our findings on the dispersal of H. baillonii to the ETP are the first confirmed report of a seagrass species crossing the American continental barrier through the Panama Canal. This highlights the potential for a similar dispersal pathway from the Caribbean into the ETP for other Halophila species that have been expanding their distributional range, such as H. stipulacea and H. ovalis.
The findings of this study clarify the phylogenetic relationships between localities, but do not indicate if these dispersal event (s) happened once or multiple times, or if a single ramet (genetic individual) or multiple ramets were transported across the Panama Canal. The establishment and maintenance of a H. baillonii population at a new location can occur by seed dispersal and germination (sexual reproduction) or by the dispersal and elongation of vegetative fragments (asexual reproduction) [36]. Halophila baillonii is a dioecious plant, whereas for sexual reproduction to occur at least one flowering shoot of each sex is needed. Female and male flowers and fruits of H. baillonii have been found in Belize [8,37] and Brazil [38,39,40]. In contrast, no fruits or seeds have been reported to date in the Pacific coast of Costa Rica for this species [12,14,15,16] and only a few reports exist of female flowers for H. baillonii in the field or grown under experimental conditions (JSV pers. obs.). Similarly, H. stipulacea is also dioecious and successfully reproduces sexually in its native region; while in its non-native regions of the Mediterranean and the Caribbean it has shown limited fruit and seed production, along with dominance of male flowering [41]. Based on these observations it is likely that H. baillonii has colonized the ETP only once and that all populations are the results of fragmentation and dispersal of the same plant. This hypothesis is supported by our data as only one haplotype was detected in our samples for the ETP. Colonisation and expansion by fragmentation has been observed before in seagrasses e.g., [42,43], and one of the most recent studies proposes that all populations of H. ovalis (previously H. johnsonii) along the eastern Florida Coast are part of the same clone [6]. To establish if H. baillonii in the ETP is a single clone a population genetic study needs to be conducted using a high number of co-dominant markers. Co-dominant markers are essential as heterozygous loci in the ETP would result in offspring with mixed allele allocations, thus confirming sexual reproduction, if it exists. Co-dominant marker loci would also assist in identifying if reproduction is only clonal, and this would be manifested by the presence of only fixed heterozygotes among all samples, as stated that is very unlikely if alleles are exchanged. Therefore, the presence of H. baillonii in other locations of the ETP needs to be assessed actively to further understand the dispersal and maintenance mechanisms of this species.
The results of this study provide knowledge that will influence the management of this seagrass species. Halophila baillonii is currently classified as Vulnerable on the IUCN Red List, given its fragmented population of limited geographic range [9,12]. The ETP populations would have been seen as highly vulnerable if they had been found to be remnants of a broader distribution pre-dating the Panama Isthmus. Furthermore, the presence of H. baillonii on the Pacific coast of Costa Rica would not lead to a reclassification of this species to the category of Least Concern. Halophila baillonii disappeared from two sites on the Pacific coast of Costa Rica [11] and thus its current status is of critical importance. At the same time, the presence of this species in the southern Pacific coast of Costa Rica has been reported by the local community dating back to 1969 [15]. The presence of this species for over 50 years indicates that conditions in the ETP are favorable for growth of H. baillonii.
Regardless of its origin, H. baillonii provides ecosystem services in the ETP and is yet to show invasive tendency by displacing native species. Halophila baillonii has been found in the ETP coexisting with Halodule beaudettei, monospecifically in deeper portions of the meadow, and as the only seagrass species at multiple locations [12,14,16]. There is evidence of grazing on H. baillonii [10] and meadows containing this species store organic carbon in their sediment on the Pacific coast of Costa Rica [12,14,15,16]. The recent introduction of H. stipulacea to the Caribbean (20 years) has led to it being considered an invasive species, as it can form extensive meadows and can outcompete native Caribbean seagrasses [41]. At other locations, H. stipulacea has quickly colonized previously unvegetated substrate and deeper areas, while it is also found growing in mixed meadows with native species [41]. Meanwhile, the introduction of H. stipulacea over 150 years ago to the Mediterranean only led to restricted and small populations [41]. Despite being an introduced species to the Mediterranean and Caribbean, where the seagrass H. stipulacea grows it changes ecosystem functioning and can provide or enhance some ecosystem services such as carbon sequestration and fish habitat [41,44,45]. At this point, we consider H. baillonii to be a recent introduction from the Caribbean to the ETP, where it is non-native and naturalized but not currently considered to be an invasive species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d15010111/s1, Supplemental Table S1: Metadata Halophila baillonii phylogenetic analysis samples; Supplemental S2: FASTA file with chloroplast reference sequences used for mapping; Supplemental S3: FASTA alignment of concatenated hybridization capture chloroplast loci; Supplemental S4: FASTA file with masked H. baillonii network alignment; Supplemental S5: Detailed chloroplast tree of H. engelmanii and H. baillonii only; Supplemental S6: H. baillonii haplotype network.

Author Contributions

Conceptualization, K.-j.v.D., M.W. and J.S.-V.; methodology, K.-j.v.D., M.W., J.S.-V., F.J.A. and E.B.; software, K.-j.v.D. and E.B.; validation, K.-j.v.D. and E.B.; formal analysis, K.-j.v.D. and E.B.; investigation, J.S.-V., J.C.C. and K.-j.v.D.; resources, J.S.-V. and M.W.; data curation, K.-j.v.D.; writing—original draft preparation, K.-j.v.D. and J.S.-V.; writing—review and editing, K.-j.v.D., J.S.-V., M.W., J.C.C., F.J.A. and E.B.; visualization, K.-j.v.D.; supervision, J.S.-V.; project administration, J.S.-V. and M.W.; funding acquisition, J.S.-V. and M.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by State Herbarium of South Australia, Botanic Gardens and State Herbarium, Department for Water and Environment, Adelaide, South Australia, Australia to conduct phylogenetic analyses. Work performed at the Universidad de Costa Rica was funded through a Fondo Semilla, grant number B9458, from the Vicerrectoría de Investigación. Collection in Brazil was funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ E26/203.002/2017) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 313698/2021-0).

Institutional Review Board Statement

Not application.

Data Availability Statement

All the short read data has been uploaded to NCBI and BioSample links can be found in Table S1.

Acknowledgments

We would like to thank Brigitta van Tussenbroek for the collection and sending of sample from Belize. We thank Jorge Cortés, Rebeca Cambronero, Jairo Moya, Héctor Arce, Gustavo Rojas, Raquel Fernández, Priscilla Montero, and Magali Marion for help with Costa Rican samples.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Nordlund, L.M.; Koch, E.W.; Barbier, E.B.; Creed, J.C. Seagrass ecosystem services and their variability across genera and geographical regions. PLoS ONE 2016, 11, e0163091. [Google Scholar] [CrossRef] [Green Version]
  2. O´Brien, K.R.; Waycott, M.; Maxwell, P.; Kendrick, G.A.; Udy, J.W.; Ferguson, A.J.; Kilminster, K.; Scanes, P.; McKenzie, L.J.; McMohon, K.; et al. Seagrass ecosystem trajectory depends on the relative timescales of resistance, recovery and disturbance. Mar. Pollut Bull 2018, 134, 166–176. [Google Scholar] [CrossRef] [Green Version]
  3. Kilminster, K.; McMahon, K.; Waycott, M.; Kendrick, G.A.; Scanes, P.; McKenzie, L.; O’Brien, K.R.; Lyons, M.; Ferguson, A.; Maxwell, P. Unravelling complexity in seagrass systems for management: Australia as a microcosm. Sci. Total Environ. 2015, 534, 97–109. [Google Scholar] [CrossRef]
  4. Short, F.; Carruthers, T.; Dennison, W.; Waycott, M. Global seagrass distribution and diversity: A bioregional model. J. Exp. Mar. Biol. Ecol. 2007, 350, 3–20. [Google Scholar] [CrossRef]
  5. Waycott, M.; McMahon, K.; Lavery, P. A Guide to Southern Temperate Seagrasses; CSIRO Publishing: Collingwood, Australia, 2014. [Google Scholar]
  6. Waycott, M.; van Dijk, K.-j.; Calladine, A.; Bricker, E.; Biffin, E. Genomics-Based Phylogenetic and Population Genetic Analysis of Global Samples Confirms Halophila johnsonii Eiseman as Halophila ovalis (R.Br.) Hook.f. Front Mar. Sci. 2021, 8, 740958. [Google Scholar] [CrossRef]
  7. Creed, J.C.; Samper-Villarreal, J. Clarification of the nomenclature of the seagrass Halophila baillonii Ascherson. Aquat. Bot. 2019, 154, 42–44. [Google Scholar] [CrossRef]
  8. van Tussenbroek, B.; Barba Santos, M.G.; Wong, J.G.R.; Van Dijk, J.K.; Waycott, M. A Guide to the Tropical Seagrasses of the Western Atlantic; Universidad Nacional Autonoma de Mexico: Mexico City, Mexico, 2010. [Google Scholar]
  9. Short, F.; Carruthers, T.; van Tussenbroek, B.; Zieman, J. Halophila baillonii. The IUCN Red List of Threatened Species 2010. Version 3.1. Available online: https://www.iucnredlist.org/species/173382/7004500 (accessed on 2 November 2022).
  10. Samper-Villarreal, J.; Bourg, A.; Sibaja-Cordero, J.A.; Cortés, J. Presence of a Halophila baillonii Asch. (Hydrocharitaceae) Seagrass Meadow and Associated Macrofauna on the Pacific Coast of Costa Rica. Pac. Sci. 2014, 68, 435–444. [Google Scholar] [CrossRef]
  11. Cortés, J. Requiem for an eastern Pacific seagrass bed. Rev Biol Trop 2001, 49 (Suppl. 2), 273–278. [Google Scholar]
  12. Samper-Villarreal, J.; Bolaños, R.C.; Heidemeyer, M.; Vargas, M.M.; Vargas, R.M. Characterization of seagrasses at two new locations in the Eastern tropical pacific (El Jobo and Matapalito, Costa Rica). Aquat. Bot. 2020, 165, 103237. [Google Scholar] [CrossRef]
  13. Samper-Villarreal, J.; Van Tussenbroek, B.I.; Cortés, J. Seagrasses of Costa Rica: From the mighty Caribbean to the dynamic meadows of the Eastern Tropical Pacific. Rev Biol Trop 2018, 66 (Suppl. 1), S53–S65. [Google Scholar] [CrossRef]
  14. Samper-Villarreal, J.; Rojas-Ortega, G.; Luis, V.-A.J.; Cortés, J. New sighting of seagrasses in the Eastern Tropical Pacific (Bahía Potrero, Costa Rica). Aquat. Bot. 2018, 151, 25–29. [Google Scholar] [CrossRef]
  15. Samper-Villarreal, J.; Cortés, J. Seagrass characterization on the southern Pacific coast of Costa Rica: History, vegetation, and environment. Bot. Mar. 2020, 63, 429–438. [Google Scholar] [CrossRef]
  16. Samper-Villarreal, J.; Moya-Ramírez, J.; Cortés, J. First characterization of seagrasses at Sámara Bay, Pacific coast of Costa Rica. Aquat. Bot. 2022, 178, 103486. [Google Scholar] [CrossRef]
  17. Cortés-Núñez, J.; Breedy-Shadid, O.; Sánchez-Noguera, C.; Pacheco-Solano, C. Los Ecosistemas Marinos del Refugio de Vida Silvestre La Flor y del Corredor Biológico Paso del Istmo, Rivas, Nicaragua; Proyecto Investigación Marino-Costero: Asegurando las Bases Científicas y Educativas para la Protección y Manejo de Tortugas Marinas en los RVS La Flor y Río Escalante-Chococente; CIMAR, Paso Pacífico, DANIDA: Managua, Nicaragua, 2012; p. 52. [Google Scholar]
  18. O’Dea, A.; Lessios, H.A.; Coates, A.G.; Eytan, R.I.; Restrepo-Moreno, S.A.; Cione, A.L.; Collins, L.S.; de Queiroz, A.; Farris, D.W.; Norris, R.D.; et al. Formation of the Isthmus of Panama. Sci. Adv. 2016, 2, e1600883. [Google Scholar] [CrossRef] [Green Version]
  19. Short, F.T.; Polidoro, B.; Livingstone, S.R.; Carpenter, K.E.; Bandeira, S.; Bujang, J.S.; Calumpong, H.P.; Carruthers, T.J.B.; Coles, R.G.; Dennison, W.C.; et al. Extinction risk assessment of the world’s seagrass species. Biol. Conserv. 2011, 144, 1961–1971. [Google Scholar] [CrossRef]
  20. Waycott, M.; van Dijk, K.-j.; Biffin, E. A hybrid capture RNA bait set for resolving genetic and evolutionary relationships in angiosperms from deep phylogeny to intraspecific lineage hybridization. bioRxiv 2021. [Google Scholar] [CrossRef]
  21. Kearse, M.; Moir, R.; Wilson, A.; Stones-Havas, S.; Cheung, M.; Sturrock, S.; Buxton, S.; Cooper, A.; Markowitz, S.; Duran, C.; et al. Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 2012, 28, 1647–1649. [Google Scholar] [CrossRef] [Green Version]
  22. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef] [Green Version]
  23. Minh, B.Q.; Schmidt, H.A.; Chernomor, O.; Schrempf, D.; Woodhams, M.D.; von Haeseler, A.; Lanfear, R. IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 2020, 37, 1530–1534. [Google Scholar] [CrossRef] [Green Version]
  24. Kalyaanamoorthy, S.; Minh, B.Q.; Wong, T.K.F.; von Haeseler, A.; Jermiin, L.S. ModelFinder: Fast model selection for accurate phylogenetic estimates. Nat. Methods 2017, 14, 587–589. [Google Scholar] [CrossRef] [Green Version]
  25. Hoang, D.T.; Chernomor, O.; von Haeseler, A.; Minh, B.Q.; Vinh, L.S. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Mol. Biol. Evol. 2017, 35, 518–522. [Google Scholar] [CrossRef]
  26. Bandelt, H.-J.; Forster, P.; Röhl, A. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 1999, 16, 37–48. [Google Scholar] [CrossRef] [PubMed]
  27. Minh, B.Q.; Nguyen, M.A.T.; von Haeseler, A. Ultrafast Approximation for Phylogenetic Bootstrap. Mol. Biol. Evol. 2013, 30, 1188–1195. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Nguyen, X.-V.; Nguyen-Nhat, N.-T.; Nguyen, X.-T.; Dao, V.-H.; Liao, L.M.; Papenbrock, J. Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region. PLoS ONE 2021, 16, e0258956. [Google Scholar] [CrossRef] [PubMed]
  29. Giachini Tosetto, E.; Bertrand, A.; Neumann-Leitão, S.; Nogueira Júnior, M. The Amazon River plume, a barrier to animal dispersal in the Western Tropical Atlantic. Sci. Rep. 2022, 12, 537. [Google Scholar] [CrossRef]
  30. Araujo, G.S.; Rocha, L.A.; Lastrucci, N.S.; Luiz, O.J.; Di Dario, F.; Floeter, S.R. The Amazon-Orinoco Barrier as a driver of reef-fish speciation in the Western Atlantic through time. J. Biogeogr. 2022, 49, 1407–1419. [Google Scholar] [CrossRef]
  31. Waycott, M.; Freshwater, D.W.; York, R.A.; Calladine, A.; Kenworthy, W.J. Evolutionary trends in the seagrass genus Halophila (Thouars): Insights from molecular phylogeny. Bull. Mar. Sci. 2002, 71, 1299–1308. [Google Scholar]
  32. Marechal, J.P.; Meesters, H.W.G.; Vedie, F.; Hellig, C. Occurrence of the alien seagrass Halophila stipulacea in Martinique (French West Indies). Mar. Biodivers. Rec. 2013, 6, e127. [Google Scholar] [CrossRef] [Green Version]
  33. Ruiz, H.; Ballantine, D.L. Occurrence of the seagrass Halophila stipulacea in the tropical West Atlantic. Bull. Mar. Sci. 2004, 75, 131–135. [Google Scholar]
  34. Lipkin, Y. Halophila stipulacea, a review of a successful immigration. Aquat. Bot. 1975, 1, 203–215. [Google Scholar] [CrossRef]
  35. Orth, R.J.; Harwell, M.C.; Inglis, G.J. Ecology of seagrasses seeds and dispersal strategies. In Seagrasses: Biology, Ecology and Conservation; Larkum, A.W.D., Orth, R.J., Duarte, C.M., Eds.; Springer: Dordrecht, The Netherlands, 2006; pp. 111–133. [Google Scholar]
  36. Kendrick, G.A.; Orth, R.J.; Statton, J.; Hovey, R.; Ruiz Montoya, L.; Lowe, R.J.; Krauss, S.L.; Sinclair, E.A. Demographic and genetic connectivity: The role and consequences of reproduction, dispersal and recruitment in seagrasses. Biol. Rev. 2017, 92, 921–938. [Google Scholar] [CrossRef] [PubMed]
  37. Short, F.T.; Fernandez, E.; Vernon, A.; Gaeckle, J.L. Occurrence of Halophila baillonii meadows in Belize, Central America. Aquat. Bot. 2006, 85, 249–251. [Google Scholar] [CrossRef]
  38. Costa, F.D.N. A Heterogeneidade Ambiental em Angiospermas Marinhas e os Efeitos Sobre a Variação Espacial da Macrofauna Associada; Universidade Federal do Ceará: Fortaleza, Brazil, 2016. [Google Scholar]
  39. Barros, K.; Costa, F.; Rocha-Barreira, C. A Halophila baillonis Ascherson bed on the semiarid coast of Brazil. Feddes Repert. 2014, 125, 93–97. [Google Scholar] [CrossRef]
  40. da Silva, N.P.; Costa, F.d.N.; Silva, M.F.S.; Mayo, S.J.; de Andrade, I.M. Seagrasses of Piauí, Brazil: A floristic treatment. Feddes Repert. 2018, 129, 43–50. [Google Scholar] [CrossRef]
  41. Winters, G.; Beer, S.; Willette, D.A.; Viana, I.G.; Chiquillo, K.L.; Beca-Carretero, P.; Villamayor, B.; Azcárate-García, T.; Shem-Tov, R.; Mwabvu, B.; et al. The tropical seagrass Halophila stipulacea: Reviewing what we know from its native and invasive habitats, alongside identifying knowledge gaps. Front Mar. Sci. 2020, 7, 300. [Google Scholar] [CrossRef]
  42. Smulders, F.O.H.; Vonk, J.A.; Engel, M.S.; Christianen, M.J.A. Expansion and fragment settlement of the non-native seagrass Halophila stipulacea in a Caribbean bay. Mar. Biol. Res. 2017, 13, 967–974. [Google Scholar] [CrossRef] [Green Version]
  43. McMahon, K.; van Dijk, K.-j.; Ruiz-Montoya, L.; Kendrick, G.A.; Krauss, S.L.; Waycott, M.; Verduin, J.; Lowe, R.J.; Statton, J.; Brown, E.; et al. The movement ecology of seagrasses. Proc. R Soc. B-Biol. Sci. 2014, 282, 20140878. [Google Scholar] [CrossRef] [PubMed]
  44. Viana, I.G.; Siriwardane-de Zoysa, R.; Willette, D.A.; Gillis, L.G. Exploring how non-native seagrass species could provide essential ecosystems services: A perspective on the highly invasive seagrass Halophila stipulacea in the Caribbean Sea. Biol. Invasions 2019, 21, 1461–1472. [Google Scholar] [CrossRef]
  45. Wesselmann, M.; Geraldi, N.R.; Duarte, C.M.; Garcia-Orellana, J.; Díaz-Rúa, R.; Arias-Ortiz, A.; Hendriks, I.E.; Apostolaki, E.T.; Marbà, N. Seagrass (Halophila stipulacea) invasion enhances carbon sequestration in the Mediterranean Sea. Global Change Biol. 2021, 27, 2592–2607. [Google Scholar] [CrossRef]
Diversity 15 00111 g002 550
Figure 2. Maximum-likelihood topology obtained from chloroplast hybridization capture of 24 chloroplast regions for 45 samples of Halophila using PhyML, Halophila beccarii was used to root the tree.
Figure 2. Maximum-likelihood topology obtained from chloroplast hybridization capture of 24 chloroplast regions for 45 samples of Halophila using PhyML, Halophila beccarii was used to root the tree.
Diversity 15 00111 g002
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MDPI and ACS Style

van Dijk, K.-j.; Waycott, M.; Biffin, E.; Creed, J.C.; Albertazzi, F.J.; Samper-Villarreal, J. Phylogenomic Insights into the Phylogeography of Halophila baillonii Asch. Diversity 2023, 15, 111. https://doi.org/10.3390/d15010111

AMA Style

van Dijk K-j, Waycott M, Biffin E, Creed JC, Albertazzi FJ, Samper-Villarreal J. Phylogenomic Insights into the Phylogeography of Halophila baillonii Asch. Diversity. 2023; 15(1):111. https://doi.org/10.3390/d15010111

Chicago/Turabian Style

van Dijk, Kor-jent, Michelle Waycott, Ed Biffin, Joel C. Creed, Federico J. Albertazzi, and Jimena Samper-Villarreal. 2023. "Phylogenomic Insights into the Phylogeography of Halophila baillonii Asch." Diversity 15, no. 1: 111. https://doi.org/10.3390/d15010111

APA Style

van Dijk, K. -j., Waycott, M., Biffin, E., Creed, J. C., Albertazzi, F. J., & Samper-Villarreal, J. (2023). Phylogenomic Insights into the Phylogeography of Halophila baillonii Asch. Diversity, 15(1), 111. https://doi.org/10.3390/d15010111

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