4.1 Species diversification
This study represents the first phylogenetic analysis of the genusToxicodryas . Phylogenetic analyses of our two-locus Sanger data
set and 2848-locus RADseq SNP data set reveal two deeply divergent,
strongly supported lineages in T. blandingii and three inT. pulverulenta (Fig. 2; Fig. S2). Although today, the two
recognized species are broadly sympatric, clades within each species are
generally situated allopatrically across river barriers. The two clades
within T. blandingii are separated either by the Sanaga River in
Cameroon or the Congo River in the DRC. Both rivers have frequently been
interpreted as population barriers in other terrestrial vertebrates
(Blackburn, 2008; Jongsma et al., 2018; Leaché et al., 2019; Leaché &
Fujita, 2010; Portik et al., 2017), but additional sampling and
comparative analyses will be needed to determine which river played the
most deterministic role in shaping genetic structure in this species. Of
the three T. pulverulenta clades, one is distributed in West
Africa (albeit with limited sampling) and two are distributed in Central
Africa, separated by the western Congo River. Our population structure
analyses are concordant with phylogenetic analyses supporting five
distinct genetic clusters (Fig. 4). Minor levels of admixture appear to
have occurred between the T. pulverulenta clades separated by the
western Congo River, and between the two clades of T. blandingiiin the sample collected at the Sanaga River (Fig. 4). In both species,
the Congo River barrier seems to be stronger in the west where the river
is wider, and the current is stronger. In the eastern DRC samples of
clades from both species can be found on either side of this river (Fig.
3)
Divergence time estimates from a time-calibrated phylogeny also fail to
reject predictions derived from the river-barrier hypothesis.Toxicodryas blandingii and T. pulverulenta diverged in the
early to mid-Miocene, and subsequent intraspecific diversification took
place in the late Miocene to the Pliocene (Fig. 2). The Congo River, a
barrier in the Central African T. pulverulenta (divergence
time: ~4.1 Mya), and a potential barrier in T.
blandingii (divergence time: ~8.6 Mya), dates back to
the mid-late Miocene (Flügel et al., 2015; Stankiewicz & de Wit, 2006).
The Sanaga River, another potential barrier in T. blandingii , has
a poorly known geological history, but likely dates back to the
formation of the Adamawa Plateau in the late Eocene-early Oligocene
(Fagny et al., 2016). Similar mid to late Miocene divergence times have
been noted for other widespread Central and West African taxa including
frogs (Bell et al., 2017; Jongsma et al., 2018; Zimkus et al., 2017),
and terrestrial snakes (Portillo et al., 2019), and similar West to
Central African distribution splits have been seen in forest cobras
(Wüster et al., 2018), frogs (Leaché et al., 2019), lizards (Allen et
al., 2019), and shrews (Jacquet et al., 2015). The Congo river has been
a well-known barrier to many species including primates (Harcourt &
Wood, 2012; Mitchell et al., 2015; Telfer et al., 2003), shrews (Jacquet
et al., 2015), and frogs (Charles et al., 2018). However, while the
timing and locations of population divergences in this study correspond
with river barriers, the Miocene was also a time of global climatic
change characterized by dramatic cooling and vegetation shifts
throughout sub-Saharan Africa (Herbert et al., 2016; Jacobs, 2004;
Menegon et al., 2014). Although most research surrounding the role of
refugia in driving diversification has focused on the dramatic climate
oscillations of the Pleistocene, it is likely that refugia are able to
form during any period of climatic change (Haffer, 1997; Hampe & Jump,
2011; Jansson & Dynesius, 2002), but the role of possible older refugia
has received little attention in the literature (Hampe & Jump, 2011).
Migration analyses support the western Congo River and the Dahomey Gap
as barriers to gene flow in the genus Toxicodryas (Fig. 5a). The
Dahomey gap is a natural savanna region in West Africa that separates
the upper and lower Guinean rainforests, and which has been previously
identified as a dispersal barrier for arboreal species (e.g. Rödel,
Emmrich, Penner, Schmitz, & Barej, 2014; Schunke & Hutterer, 2005).
Both areas also support lower genetic diversity in Toxicodryasthan expected under a pure isolation-by-distance model (Fig. 5b),
emphasizing the biological reality of this barrier for
forest-associated, primarily arboreal vertebrates despite the fact that
both Toxicodryas species have been found in forest patches within
the Dahomey gap. Our demographic analyses further suggest that riverine
dispersal barriers between clades are strong, indicating divergence
without gene flow between the two T. blandingii clades and
divergence with minor gene flow across the Congo River in the two
Central African T. pulverulenta clades (Fig. 6). Contemporary
gene flow was ruled out with high confidence in both species (Table S3).
In light of the Miocene divergence times and lack of gene flow between
these five clades, it is likely that they represent distinct
evolutionary lineages and, thus, surveys of morphological data and
analyses of phenotypic variation are underway to determine if formal
taxonomic revision is justified.