MOLECULAR CLOCK DATING OF SÃO TOMÉ AND PRÍNCIPE FLORAL ENDEMICS:A CASE STUDY IN AFROCARPUS BY | ALEX GOKE ABSTRACT 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. INTRODUCTION | This information will assist in evaluating the status of at- Afrocarpus (Buchanan-Hamilton & N.E.Gray) C.N.Page risk species and distributions in the region and highlight the is a poorly-described genus of two to six tree species of the importance of understanding diversification histories as they family Podocarpaceae (Pinales) [1, 2]. Assumed to be of relate to conservation. Pleistocene origin, members of the genus are characteristic of the distinctive Afromontane floristic region which METHODS AND MATERIALS | ------HH---- includes over 4000 species, nearly three-quarters of which VISUALIZING DISTRIBUTION OF AFROCARPUS ---- are endemic. [3, 4]. Afrocarpus is restricted to the eastern Occurrence records for Afrocarpus were obtained on 2 and southern high-elevation forests of the African continent March Occurrence records for Afrocarpus were obtained with the exception of A. mannii (Hook.f.) C.N.Page and A. on 2 March 2015 from the Global Biodiversity Information gaussenii Woltz C.N.Page, which are found endemic to the Forum [7]. One hundred and ninety-one georeferenced island of São Tomé off the coast of Equatorial Guinea and occurrences were available, representing all species except eastern Madagascar, respectively [3]. A. gaussenii. Distributions were visualized within the DIVA- A. mannii occurs only on the upper slopes (1,300m+) GIS platform and then generalized in Photoshop CC v. 2014. REPORTS of mountainous Pico de São Tomé, confined to a mere Distribution of A. gaussenii was inferred from the literature. 25km2 area of cloud forest where it faces eradication PHYSICAL AND BIOLOGICAL DESCRIPTION OF SÃO through deforestation and stochastic events. These threats TOMÉ AND PRÍNCIPE | have earned the population a “Vulnerable” designation on A literature review was conducted for information relevant the International Union for Conservation of Nature Red to the biological and geological history of the island nation of List of Threatened Species [5]. It is the only gymnosperm São Tomé and Príncipe, as well as the larger Gulf of Guinea representative on the island [6]. region. Specifically, conclusions about the geologic origins of The continental members of the genus, including A. dawei the island, its spatial relations to mainland Africa through (Stapf) C.N.Page, A. falcatus, A. gracilior (Pilg.) C.N.Page, time, the modern biotic structure of the island, and factors and A. usambarensis (Pilg.) C.N. Page, maintain stable influencing biodiversity, including regional aeolian and populations throughout the scattered Afromontane regions fluvial pollen circulation, were researched. Principal findings stretching from South Africa to Ethiopia [5]. from the review were incorporated in this study in order to With this study, I seek to describe the timing of diversification contextualize phylogenetic results. in Afrocarpus that led to the eventual establishment of an PHYLOGENETIC ANALYSIS | ----- endemic A. mannii population over 250km from continental Partial sequences of the maturase K (matK) gene, 5.8S Africa and 2,400km from the rest of the genus. Molecular ribosomal RNA gene, 26S ribosomal RNA gene, and ribulose- clock dating will grant insight into the diversification 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) history of Afrocarpus, and not only will it aid in explaining gene were obtained for the family Podocarpaceae, and contemporary spatial patterns of diversity in the genus, but of NEEDLY and phytochrome P (PHYP) gene partial sequences São Tomé and Príncipe as a whole. 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 JUST Journal | Volume 1 PAGE 29 JUST-2016_Final.indd 29 4/5/2016 4:25:40 PM MOLECULAR CLOCK DATING OF SÃO TOMÉ AND PRÍNCIPE FLORAL ENDEMICS:A CASE STUDY IN AFROCARPUS Appendix I [12, 13]. VISUALIZING DISTRIBUTION OF AFROCARPUS | Genetic sets containing multiple data for any one taxon were Figure 1 depicts the continental and oceanic distribution reduced in an arbitrary fashion as to contain only one sequence of Afrocarpus. As shown, A. mannii exists solely on the per species. Sequences were then aligned via MAFFT v. 7.017 main island of São Tomé and Príncipe, and approximately using an auto alignment algorithm and 200PAM/k=2 scoring 2,400km from the rest of the genus. A. dawei persists in the matrix with a 1.53 gap open penalty and a 0.123 offset value highlands of East-Central Africa, circumnavigating Lake within Geneious v. 8.1.4 [14-16]. Aligned sequences were Victoria through Uganda, Tanzania, and Kenya. A. falcatus then concatenated in Geneious and delivered to the CIPRES is located mainly in South Africa, though extends somewhat Science Gateway v. 3.3 on 28 April 2015 for phylogenetic into Southern Mozambique with a disjunct path through tree inference using RAxML-HPX Blackbox algorithm v. central Tanzania. A. gaussenii, as previously stated, is isolated 8.1.11 and run for two hours with default input parameters to the montane eastern regions of Madagascar. A. gracilior [17, 18]. The output was reduced so that 162 taxa (including maintains a similar distribution pattern to A. dawei, though select Podocarpaceae, Araucariaceae, and Cupressaceae extends further north into Ethiopia. Finally, A. usambarensis as outgroup) were retained for analysis from 305 to reduce inhabits the gap between recorded A. dawei, A. falcatus, processing time and to focus results (Appendix I). 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]. FIG. 1 | Contemporary distribution of Afrocarpus across São Tomé has never made contact with mainland Africa continental and oceanic Africa. or other oceanic CVL land-masses. The relative distance Evolutionary rate of the best maximum likelihood/rapid between masses and depth of the ocean that surrounds them bootstrapping result tree was assessed with reference to (ca. 1,800m) prevented land bridges from forming during estimated divergences of Microcachrys tetragona (x=23.5 historical glaciation events [28]. MYA, s=1), Nageia (x=47 MYA, s=4), and Cupressaceae São Tomé has a constituent flora of 602 species, of which (x=245 MYA, s=15) [19-21]. Parameters for analysis were set 96, including an entire genus, are endemic [29]. Other in BEAUTi v. 1.7.5 then delivered to CIPRES for Bayesian estimates have placed rates of endemism as high as 20% [30]. analysis using BEAST v. 1.8.0. Ten million generations Montane elements are particularly prone to endemism [31]. were evaluated and recorded every 1,000 generations under Between the CVL oceanic islands, only 16 species are shared, REPORTS GTR, Yule speciation, nucleotide substitution models, indicating each island received its biota independently from and a log-normal relaxed clock algorithm [22-24]. The each other [28]. The recognized uniqueness of the biota has posterior