Introduction
Identifying the driving forces promoting speciation is a key question in
evolutionary biology (Mayr, 1942). Species pair with very low
morphological differentiation but very significant genetic divergence
(and vice versa ) offers excellent models for evolutionary
biologists, which has improved our understanding of how species
originate. Generally, limited gene flow is believed to hinder genetic
divergence and lead to speciation (Rifkin, Castillo, Liao, & Rausher,
2019). However, researchers have focused on several pairs of closely
related species diverged with gene flow (Aguirreliguori et al., 2019;
Cooper, Whittall, Hodges, & Nordborg, 2010; Rifkin et al., 2019; Wu,
Wang, Wang, Shrestha, & Liu, 2022; Zhao, Yin, Pan, & Gong, 2018; X. M.
Zheng & Ge, 2010). Restricted gene flow fosters genetic and phenotypic
divergence that may cause speciation (D. Schluter, 2001; Slatkin, 1987).
The geographic isolation is generally considered the major driving force
that impedes gene flow and promotes speciation, meanwhile, local
adaptation can also generate barriers to gene flow and lead to
ecological speciation (Aguirreliguori et al., 2019; Nosil, 2012; Rundle
& Nosil, 2005; D Schluter & Conte, 2009). In addition, it is
mysterious that some species pairs are morphologically similar while can
be recognized using molecular markers (i.e. cryptic species)(Bickford et
al., 2007). Comparing patterns of intra- and inter-specific genetic
differentiation between morphological similarity species should be of
great interest for understanding the process of their speciation.
Cycads, the most primitive living seed plants, include two families
(Cycadaceae and Zamiaceae) with around 360 species of ten genera
(Calonje, Stevenson, & Stanberg, 2022; Christenhusz et al., 2011).
Although the cycad lineage is ancient, the extant cycad species have
evolved recently with a synchronous global diversification during the
late Miocene and are not older than 12 million years (Xiu Yan Feng, Liu,
Chiang, & Gong, 2017; X. Y. Feng, Zheng, & Gong, 2016; Nagalingum et
al., 2011; S. Y. Xiao, Ji, Liu, & Gong, 2020; Y. Zheng, Liu, & Gong,
2016). However, a recent study combining plastid phylogenomic, plate
tectonic and fossil evidence indicated that the diversification of
Cycadaceae (~69-43 million years ago, Palaeogene) was
not that young (Jian Liu, Lindstrom, Marler, & Gong, 2022). The genusCycas L., the sole representative of the family Cycadaceae,
occurs in the Australia, India, Malesian region, Japan and Southeast
Asia, extending to Micronesia and Polynesia, Madagascar and East Africa
(Jones, 2002). Biogeographic analyses strongly favor a South China
origin for the living Cycas and an early dispersal to Indochina
(L. Q. Xiao & Moller, 2015). However, controversial views emerged
later. The ancestral area of Cycadaceae was recently inferred as a
tropical region (i.e. Indochina) (Mankga, et al., 2020) or East Asia
(Liu, et al., 2021). Cycas species have the presence of a crown
of bird’s feather-like leaves, and their leaf bases all around the stem,
which makes them look like palms in appearance. Two types of pinnae are
presented in this genus, namely, pinnae simple and pinnae dichotomously
divided. Most Cycas species are pinnae simple and only fourCycas species have dichotomously divided pinnae (Ken D. Hill,
Nguyen, & Loc, 2004). Based on the key (Appendix S1) and morphological
description (Ken D. Hill et al., 2004), C. bifida and C.
micholitzii are the most two difficult sister species to distinguish in
morphology (Figure S1), whereas their phylogenetic relationships are
controversial (J. Liu et al., 2018; Y. Liu et al., 2022). According to
the phylogeny of DNA fragment data, the two species had great genetic
differentiation (J. Liu et al., 2018), while based on the transcriptome
data, they showed sister species relationship (Y. Liu et al., 2022).
Cycas bifida is most closed with C. micholitzii but has
larger male cones with microsporophyll tips that are generally without
spines. Despite its original name (Appendix S1), the species has no
affinity with C. rumphii . Geographically, C. bifida occurs
in southwest China and the northern Vietnam, while C. micholitziioccurs in the Annam Highlands region of central Vietnam, southeast Laos
and the northeast Cambodia (Ken D. Hill, 2008; Ken D. Hill et al.,
2004). Their distribution areas are separated by the Red River Fault
Zone (RRFZ) which is considered as a biogeographical barrier forCycas species distributed in this area (Jian Liu, Zhou, & Gong,
2015; Tang, 2004; Yang, Feng, & Gong, 2017; Y. Zheng et al., 2016). The
RRFZ, defined by South China in the northeastern and Indochina in the
southwestern, is proposed to be formed by India-Asia collision (Searle,
2006). For years, people have been debating it experienced left-lateral
strike-slip in the first 30 - 16 Ma of collision (Tapponnier, Peltzer,
Le Dain, Armijo, & Cobbold, 1982) with at least 15 km of displacement
(Schoenbohm, Burchfiel, Liangzhong, & Jiyun, 2006) and presently
undergone right-lateral motion since the late Neogene (8 - 7 Ma) with
lower slip rate (Leloup et al., 1993; Schoenbohm, Burchfiel, &
Liangzhong, 2006; Zuchiewicz & Cuong, 2009).
In the study reported here, we are aiming to find out the driving forces
for these two species divergence by using multidisciplinary methods. We
firstly used molecular approach to estimate the degree of species
divergence among C. bifida and C. micholitzii . Then by
integrating coalescent-based analysis (IMa), isolation by distance
analysis (IBD), Barrier analysis, and Niche consistency detection, we
tested the following specific hypotheses for how present patterns of
genetic divergence arose: (i) they originated from the same ancestor or
were one species before, and significant divergence between the twoCycas species was due to the long-term geographical isolation;
(ii) they were originated from different ancestors. Their morphological
similarities, especially the bifurcations of the pinnae, are a
phenomenon of latter convergent evolution because of similar ecological
niche.