Molecular Clock Dating of São Tomé and Príncipe Floral Endemics: A Case Study in Afrocarpus

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The island nation of São Tomé and Príncipe in the Gulf of Guinea hosts an incredible diversity of floral species relative to its size and distance from larger land masses. The occurrence of Afrocarpus mannii (Hook.f.) C.N.Page on São Tomé, in particular, illustrates a classic example of long distance dispersal from the continental distribution of the rest of the African genus, providing a unique opportunity to study diversification patterns in a disjunct Afromontane population. Phylogenetic analysis of Afrocarpus is carried out using numerous publicly available genomic sequences. Molecular clock dating indicates a time of divergence for A. mannii at approximately 3.5 MYA. The findings presented here suggest a pre-Pleistocene speciation event discordant with historical assumptions. Further study will likely reveal similar diversification patterns for other island endemics. These data support the need for conservation action to protect a rare and informative species in one of the most biologically diverse regions of the planet. Afrocarpus mannii at approximately 3.5 MYA (1.2996-8.6183 95% HPD) well after the volcanic formation of São Tomé and Principe. Lack of extant Afrocarpus on nearby mainland habitat implies an extreme long- distance dispersal event towards establishment on the island. The findings presented here indicate a previously undocumented and extremely rare dispersal event to one of the most biologically diverse, yet often overlooked, regions of the planet.

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Introduction

Afrocarpus (Buchanan-Hamilton & N.E.Gray) C.N.Page is a poorly-described genus of two to six tree species of the family Podocarpaceae (Pinales) [1, 2]. Assumed to be of Pleistocene origin, members of the genus are characteristic of the distinctive Afromontane floristic region which includes over 4000 species, nearly three-quarters of which are endemic. [3, 4]. Afrocarpus is restricted to the eastern and southern high-elevation forests of the African continent with the exception of A. mannii (Hook.f.) C.N.Page and A. gaussenii Woltz C.N.Page, which are found endemic to the island of São Tomé off the coast of Equatorial Guinea and eastern Madagascar, respectively [3].

A. mannii occurs only on the upper slopes (1,300m+) of mountainous Pico de São Tomé, confined to a mere 25km2 area of cloud forest where it faces eradication through deforestation and stochastic events. These threats have earned the population a “Vulnerable” designation on the International Union for Conservation of Nature Red List of Threatened Species [5]. It is the only gymnosperm representative on the island [6]. The continental members of the genus, including A. dawei (Stapf) C.N.Page, A. falcatus, A. gracilior (Pilg.) C.N.Page, and A. usambarensis (Pilg.) C.N. Page, maintain stable populations throughout the scattered Afromontane regions stretching from South Africa to Ethiopia [5]. With this study, I seek to describe the timing of diversification in Afrocarpus that led to the eventual establishment of an endemic A. mannii population over 250km from continental Africa and 2,400km from the rest of the genus. Molecular clock dating will grant insight into the diversification history of Afrocarpus, and not only will it aid in explaining contemporary spatial patterns of diversity in the genus, but of São Tomé and Príncipe as a whole. This information will assist in evaluating the status of at-risk species and distributions in the region and highlight the importance of understanding diversification histories as they relate to conservation.

Methods and Materials

Visualizing Distribution of Afrocarpus

Occurrence records for Afrocarpus were obtained on 2 March Occurrence records for Afrocarpus were obtained on 2 March 2015 from the Global Biodiversity Information Forum [7]. One hundred and ninety-one georeferenced occurrences were available, representing all species except A. gaussenii. Distributions were visualized within the DIVA-GIS platform and then generalized in Photoshop CC v. 2014. Distribution of A. gaussenii was inferred from the literature.

Physical and Biological Description of São Tomé and Príncipe

A literature review was conducted for information relevant to the biological and geological history of the island nation of São Tomé and Príncipe, as well as the larger Gulf of Guinea region. Specifically, conclusions about the geologic origins of the island, its spatial relations to mainland Africa through time, the modern biotic structure of the island, and factors influencing biodiversity, including regional aeolian and fluvial pollen circulation, were researched. Principal findings from the review were incorporated in this study in order to contextualize phylogenetic results.

Phylogenetic Analysis

Partial sequences of the maturase K (matK) gene, 5.8S ribosomal RNA gene, 26S ribosomal RNA gene, and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene were obtained for the family Podocarpaceae, and NEEDLY and phytochrome P (PHYP) gene partial sequences were obtained for the order Pinales [8-11]. Sequences were selected for reliability and ease of acquisition. GenBank accession numbers for all taxa evaluated can be found in Appendix I [12, 13]. Genetic sets containing multiple data for any one taxon were reduced in an arbitrary fashion as to contain only one sequence per species. Sequences were then aligned via MAFFT v. 7.017 using an auto alignment algorithm and 200PAM/k=2 scoring matrix with a 1.53 gap open penalty and a 0.123 offset value within Geneious v. 8.1.4 [14-16]. Aligned sequences were then concatenated in Geneious and delivered to the CIPRES Science Gateway v. 3.3 on 28 April 2015 for phylogenetic tree inference using RAxML-HPX Blackbox algorithm v. 8.1.11 and run for two hours with default input parameters [17, 18]. The output was reduced so that 162 taxa (including select Podocarpaceae, Araucariaceae, and Cupressaceae as outgroup) were retained for analysis from 305 to reduce processing time and to focus results (Appendix I).

FIG. 1 | Contemporary distribution of Afrocarpus across continental and oceanic Africa.

FIG. 1 | Contemporary distribution of Afrocarpus across
continental and oceanic Africa.

Evolutionary rate of the best maximum likelihood/rapid bootstrapping result tree was assessed with reference to estimated divergences of Microcachrys tetragona (x=23.5 MYA, s=1), Nageia (x=47 MYA, s=4), and Cupressaceae (x=245 MYA, s=15) [19-21]. Parameters for analysis were set in BEAUTi v. 1.7.5 then delivered to CIPRES for Bayesian analysis using BEAST v. 1.8.0. Ten million generations were evaluated and recorded every 1,000 generations under GTR, Yule speciation, nucleotide substitution models, and a log-normal relaxed clock algorithm [22-24]. The posterior distribution was extracted and summarized using TreeAnnotator v. 1.8.2 and then visualized with FigTree v.1.7.4.

Visualizing Distribution of Afrocarpus

Figure 1 depicts the continental and oceanic distribution of Afrocarpus. As shown, A. mannii exists solely on the main island of São Tomé and Príncipe, and approximately 2,400km from the rest of the genus. A. dawei persists in the highlands of East-Central Africa, circumnavigating Lake Victoria through Uganda, Tanzania, and Kenya. A. falcatus is located mainly in South Africa, though extends somewhat into Southern Mozambique with a disjunct path through central Tanzania. A. gaussenii, as previously stated, is isolated to the montane eastern regions of Madagascar. A. gracilior maintains a similar distribution pattern to A. dawei, though extends further north into Ethiopia. Finally, A. usambarensis inhabits the gap between recorded A. dawei, A. falcatus, and A. gracilior populations through northern Tanzania. Afrocarpus has not been recorded in West Africa [3].

Physical and Biological Description of São Tomé and Príncipe

São Tomé is the larger of the two oceanic islands of São Tomé and Príncipe. It rises ca. 5,000m from the ocean floor in the Gulf of Guinea, providing 964km2 (with Príncipe) of habitable surface. The island is one of four that constitutes the oceanic sector of the Cameroon Volcanic Line (CVL), which extends from the southern island of Annobón to the plateaus of Eastern Nigeria/Northern Cameroon [25].

The exact timing of formation of the CVL oceanic islands is a contentious issue. Competing theories place formation from between 66 to 80 MYA and 124 to 140 MYA [26]. Discussion of these debates is beyond the scope of this study, and therefore all theories seemed rational to consider in analysis. While the origins of the volcanic chain are still being debated, the fact that São Tomé is of volcanic origin is authoritative, as the island remains volcanically active. Argon isochrome sampling indicates lava flow as recently as 0.03 MYA [27]. São Tomé has never made contact with mainland Africa or other oceanic CVL land-masses. The relative distance between masses and depth of the ocean that surrounds them (ca. 1,800m) prevented land bridges from forming during historical glaciation events [28].

São Tomé has a constituent flora of 602 species, of which 96, including an entire genus, are endemic [29]. Other estimates have placed rates of endemism as high as 20% [30]. Montane elements are particularly prone to endemism [31]. Between the CVL oceanic islands, only 16 species are shared, indicating each island received its biota independently from each other [28]. The recognized uniqueness of the biota has earned the island World Wildlife Foundation “Center of Plant Diversity” designation, though only 35% of the 50km2 forest is protected to some degree and continues to be encroached upon by expanding plantation activity [31, 32].

Regional dispersal events to the island are rare. Movement of pollen into the Gulf from extant podocarps in the western forests of Cameroon represents less than 1% of grain deposition. On a larger scale, long-distance dispersal to the Gulf of Guinea of eastern and southern African species, such as continental Afrocarpus, has only been observed through Asteraceae Tubuliflorae and Combretaceae-Melastomataceae pollen grains, which represent minor contributions to the marine sediment pollen bank of the Gulf [33, 34]. Afrocarpus as a whole is considered to be severely dispersal-limited [3]. Together, these factors make recent or continued dispersal events of Afrocarpus highly unlikely, particularly in the face of prevailing south-westerly monsoon winds [35].

FIG. 2 | Divergence in interior Pinales (red bars indicate 95% HDP interval at ancestral nodes, estimated ages available in Appendix I).

FIG. 2 | Divergence in interior Pinales (red bars indicate 95% HDP interval at ancestral nodes, estimated ages available in Appendix I).

Phylogenetic Analysis

Bayesian analysis generated trees that substantially conform to those presented by larger studies [e.g. 8-11, 36, 37]. The best phylogenetic estimate is shown in Figure 2. Molecular clock analyses of Afrocarpus yielded divergence times of ancestral node to A. mannii at 3.5 million years ago (MYA) with a 95% highest posterior density interval of 1.3 to 8.6 MYA (Fig. 3). Complete divergence data are listed in Appendix I.

Discussion

São Tomé, Príncipe, and other Gulf of Guinea island masses are largely agreed to be of volcanic origin, and with no land bridges forming between either island and continental Africa, their constituent flora are entirely the result of long distance dispersal or human-mediated establishment. Time of divergence for A. mannii is established at 3.49 MYA, well before the beginning of the Pleistocene. Other members of the genus appear more recently from 2.8 to 0.7 MYA. These data suggest repetitive subjection of a non-diversifying A. manni lineage to habitat fragmentation and continental aridification throughout the Quaternary, while other members underwent further diversification as assumed through the Pleistocene. This would seem to indicate pre-Pleistocene dispersal to São Tomé where A. mannii continues to persist, supporting in part the standing hypothesis of an “old” Afrocarpus distribution [3].

FIG. 3 | Divergence in higher Podocarpoids (red bars indicate 95% HPD interval at ancestral nodes, values indicate mean age).

FIG. 3 | Divergence in higher Podocarpoids (red bars indicate 95% HPD interval at ancestral nodes, values indicate mean age).

The resolution of these data are limited by the number and confidence of fossil reference taxa and completeness of molecular sequences. Further analysis with additional sequence and reference data would likely corroborate these findings and strengthen our understanding of this vastly understudied genus. Additionally, diversification patterns in Afrocarpus, being the only gymnosperm representative on the island, may not be congruent with those of other regional flora as suggested, though a few studies in to angiosperm groups have shown similar results [e.g. 38].

Knowledge of the biogeographical history of São Tomé and Príncipe endemic flora, particularly that of Afrocarpus mannii, has far-reaching implications in the contemporary understanding of patterns in Afromontane diversification and distribution resulting in regions of endemic diversity. Specifically, the findings here invoke theory of Afromontane pre-Pleistocene paleobiotic movement and diversification events and confirm the merit of phylogenetics in reaching such conclusions. Additionally, awareness of the severity of endemism in the region, coupled with the rarity of long-distance dispersal events, such as the one introducing Afrocarpus to the island, can inform and direct conservation efforts, knowing that rescue effects will not support the local population in the face of land use change, exploitation activities, and climate change effects that threaten the biodiversity of the island. Further study of the flora is needed to complement the findings provided here in order to support the immediacy of action needed to conserve one of the most unique and diverse biological hotspots in the world.

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