1. INTRODUCTION
For more than two hundred years, scientists have pondered the most
pervasive pattern in biogeography, the latitudinal diversity gradient, a
striking pattern of increasing species diversity from the poles toward
the equator (Allen & Gillooly, 2006; Darlington, 1957; Darwin, 1859;
Hutchinson, 1959; Jablonski, Roy, & Valentine, 2006; Pianka, 1966;
Ricklefs & Schluter, 1993; Rohde, 1992; Rosenzweig, 1995; Wallace,
1878). Potential explanations of this pattern have been provided from
diverse perspectives, including that tropical regions have a wider array
of niches (Buckley et al., 2010; Lamanna et al., 2014; Stevens, 2011),
higher primary productivity (Hawkins, Porter, Diniz-Filho, & Alexandre,
2003; Jetz & Fine, 2012; Whittaker, Nogués‐Bravo, & Araújo, 2007),
higher environmental heterogeneity (Janzen, 1967; Stein, Gerstner, &
Kreft, 2014; Stevens, 1989), greater land area (Fine & Ree, 2006;
Rosenzweig, 1995; Terborgh, 1973), and higher climatic stability
(Harrison & Noss, 2017; Hawkins, Diniz-Filho, Jaramillo, & Soeller,
2007). In more recent studies of historical tropical biogeography,
researchers have focused on speciation processes in major tropical
rainforests to explain their high biodiversity (e.g. Cardillo, Orme &
Owens, 2005; Jablonski et al., 2006; Ricklefs, 2006; Smith et al., 2017;
Weir & Schluter, 2007; Wiens & Donoghue, 2004; Wiens, Sukumaran,
Pyron, & Brown, 2009).
Three major allopatric diversification mechanisms have been proposed in
the classical literature to explain species diversity in the Amazon: the
“river hypothesis” in which species and populations diverged across
river barriers (Ayres & Clutton-Brock, 1992; Bates, 1863; Hershkovitz,
1977; Mayr, 1942; Sick, 1967; Wallace, 1853); the “refuge hypothesis”
in which forests fragmented during Earth’s cold or dry climate cycles
(i.e. the Pleistocene glaciation cycles), causing isolation and
divergence in small forest patches (Haffer, 1969, 1974, 1982; Prance,
1982; Vanzolini, 1973; Vanzolini & Williams, 1970); and an amalgamate
“river-refuge hypothesis” in which speciation was promoted by a
combination of river barriers and climate driven vegetation changes
(Ayres & Clutton-Brock, 1992; Haffer 1992, 1993). These hypotheses have
been widely used in the study of Neotropical biodiversity and the
mechanisms of its production (e.g. Gascon et al., 2000; Haffer, 2008;
Patton & Silva, 2005; Richardson, Pennington, Pennington, &
Hollingsworth, 2001; Weir, 2006). However, because the early scientific
focus was primarily on the Amazon (Amorim, 1991; Cracraft, 1985;
DeMenocal, 2004; Haffer, 1969, 1997; Plana, 2004; but see Fjeldså, 1994;
Mayr & O’Hara, 1986), and given political instability in tropical
Africa (Greenbaum, 2017; Siddig, 2019; Tolley et al. 2016), rigorous
testing of the predictions stemming from these hypotheses has been
neglected for the West and Central African rainforests until only
recently.
Based on pollen core records (Brenac, 1988; Bonnefille & Riollet, 1988;
Girese, Maley, & Brenac, 1994; Maley, 1987, 1989, 1991; Maley &
Brenac, 1987; Maley & Livingstone, 1983; Sowunmi, 1991) and species
distribution data (Colyn, 1987, 1991; Rietkerk, Ketner, & De Wilde,
1995; Richards, 1963; Sosef, 1991), Maley (1996) proposed several
rainforest refugia for sub-Saharan Africa that are still widely used
today (e.g. Bell et al., 2017; Hughes, Kusamba, Behangana, & Greenbaum,
2017; Huntley, Castellanos, Musher, & Voelker, 2019; Jongsma et al.,
2018; Larson, Castro, Behangana, & Greenbaum, 2016; Penner, Wegmann,
Hillers, Schmidt & Rödel, 2011, Portik et al. 2017; Fig. 1). Many of
these hypothesized refugia are located in highland areas (e.g., the
Cameroon Volcanic Line and the Albertine Rift), however, a major fluvial
refuge, located in the gallery forests around the Congo River, has been
supported by pollen core data (Maley, 1996), and distributional patterns
of multiple bird (Huntley, Harvey, Pavia, Boano, & Voelker, 2018;
Levinsky et al., 2013), mammal (Colyn, Gautier-Hion, & Verheyen, 1991;
Levinsky et al., 2013) and plant taxa (Robbrecht, 1996).
Major river barriers in West and Central Africa include the Volta, the
Sanaga, the Ogooué, the Congo, the Niger and the Cross Rivers (Fig. 1).
The exact ages of many of these rivers are unknown but are generally
estimated to date back to the Late Mesozoic to the Early Cenozoic
(80–35 mya; Goudie, 2005; Stankiewicz & de Wit, 2006). However, while
the Congo basin is quite old (Flügel, Eckardt, & Cotterill, 2015;
Stankiewicz & de Wit, 2006), the present course of the Congo River
appears to be much younger, dating to the mid to late Miocene and
corresponding to the uplift of the East African Rift (Flügel, et al.,
2015; Stankiewicz & de Wit, 2006).
Numerous phylogeographic studies have supported the importance of
rivers, refugia, or both as drivers of diversification across disparate
plant and animal species. Rivers alone have been shown to be important
barriers for some species of primates (Mitchell et al., 2015; Telfer et
al., 2003), shrews (Jacquet et al., 2015), and frogs (Charles et al.,
2018; Penner et al. 2011; Penner, Augustin & Rödel, 2019; Wieczorek,
Drews & Channing, 2000; Zimkus, Hillers & Rödel, 2010), but do not
appear to represent an important barrier for many plant species (Dauby
et al., 2014; Debout, Doucet, & Hardy, 2011; Hardy et al., 2013; Ley et
al., 2014; Lowe, Harris, Dormontt, & Dawson, 2010). Refugia are
suggested to have played an important role in the diversification of
rodents (Bohoussou et al., 2015; Nicolas et al., 2011; Nicolas, Missoup,
Colyn, Cruaud, & Denys, 2012), primates (Clifford et al., 2004; Haus et
al., 2013; Tosi, 2008), frogs (Bell et al., 2017; Jongsma et al., 2018),
lizards (Allen, Tapondjou, Greenbaum, Welton, & Bauer, 2019; Leaché et
al., 2017), birds (Fjeldså & Bowie, 2008), pangolins (Gaubert et al.,
2016), and rainforest plants (Born et al., 2011; Budde,
González-Martínez, Hardy, & Heuertz, 2013; Daïnou et al., 2010; Dauby,
Duminil, Heuertz, & Hardy, 2010; Duminil et al., 2015; Faye et al.,
2016; Gomez et al., 2009; Hardy et al., 2013; Ley et al., 2014; Ley,
Heuertz, & Hardy, 2016; Lowe et al., 2010). In some cases, divergence
patterns match both refugial and riverine predictions (Anthony et al.,
2007; Barej et al., 2011; Bohoussou et al., 2015; Gonder et al., 2011;
Jacquet et al., 2014; Jongsma et al., 2018; Leaché et al., 2019; Leaché
& Fujita, 2010; Marks, 2010; Portik et al., 2017), suggesting that both
may have played roles simultaneously—or in combination—in
evolutionary diversification. However, because of the spatial overlap of
refugia with montane and riverine systems (Hofer, Bersier, & Borcard,
1999, 2000), and the sparse pollen core and fossil records for the
tropics (Colinvaux, De Oliveira, Moreno, Miller, & Bush, 1996; Maley &
Brenac, 1998), distinguishing between these three hypotheses has been
difficult, especially when relying on phylogeographic data alone.
The three major allopatric diversification hypotheses make the following
predictions regarding species diversification patterns in tropical
African forests (1) river hypothesis: boundaries between population
distributions should correspond to riverine barriers and the ages of
populations should be relatively old, corresponding to the ages of the
rivers; (2) refuge hypothesis: population distributions should be
concordant with locations of hypothesized rainforest refugia during
cold, dry periods and populations are predicted to be relatively young,
possibly corresponding to the Pleistocene glaciation cycles; (3)
river-refugia hypothesis: population distributions should be correlated
with the locations of rainforest refugia and bounded by rivers barriers,
or will have been confined to refugial locations and additionally
subdivided by rivers. Finally, the timing of population splits should
correspond to ages of rivers but would be expected to show patterns of
expansion and contraction dating to the Pleistocene.
In this study, we use the snake genus Toxicodryas as a model
system to test the predictions of these hypotheses. The genusToxicodryas consists of two large, rear-fanged, venomous West and
Central African species, T. blandingii and T.
pulverulenta. The taxonomic placement of this genus is uncertain. They
were originally placed in the Asian genus Boiga (Schmidt, 1923),
and some authors still classify them as such, but recent phylogenetic
analyses recover them as the sister genus to the African egg eating
snakes, Dasypeltis , albeit with weak support (Pyron et al.,
2013). Both species in this genus are primarily arboreal, feeding mainly
on birds, bats, frogs and chameleons (Akani, Barieenee, & Luiselli,
1998; Chippaux & Jackson, 2019; Nagy et al., 2011; Spawls, Howell,
Hinkel, & Menegon, 2018). Because of their general arboreality, these
species are predicted to have distributions strongly correlated with
forest distribution. In addition, Toxicodryas is widely
distributed within the Congo River fluvial system and broadly across
West and Central Africa, making this genus a suitable system for testing
the competing predictions of the river, refugia and river-refugia
hypotheses.
Recent advances in paleo-climate modeling and genome-scale DNA
sequencing have opened new avenues to testing classic hypotheses of
tropical rainforest speciation (Bell et al., 2017; Leaché et al., 2019;
Portik et al., 2017). In this study, we integrate dated phylogeographic
inference, population structure analyses, and machine learning-based
demographic modeling to identify the timing of divergence as well as the
location and permeability of past and present dispersal barriers. These
genetic data are combined with paleo-distribution and climate stability
modeling to determine the congruence of historical distributions with
the refugial and river-refugial hypotheses. Our results demonstrate
that, although population distributions alone could be congruent with
any of the three hypotheses, diversification times predate the
Pleistocene, a finding that aligns with predictions of the river-barrier
hypothesis. Historical demographic analyses support models of no
migration among populations since the time of divergence, and migration
analyses suggest that the western Congo River represents one of the
strongest barriers to recent dispersal. Species paleo-distribution and
climate stability modelling show no suggestion of suitable habitat
contraction during or since the Pleistocene, allowing us to soundly
reject the predictions of refugia hypotheses in favor of the prevailing
role of riverine barriers in shaping, structuring, and maintaining
diversity in this generally arboreal, forest-associated group of endemic
African snakes.