Conservation Genetics

https://doi.org/10.1007/s10592-019-01154-8

RESEARCH ARTICLE

Population genetics, speciation, and hybridization in Dicerandra

(Lamiaceae), a North American Coastal Plain endemic,
and implications for conservation
Adam C. Payton1,2 · Andre A. Naranjo1,2 · Walter Judd1,2 · Matthew Gitzendanner2 · Pamela S. Soltis1 ·
Douglas E. Soltis1,2

Received: 11 September 2018 / Accepted: 5 February 2019

© Springer Nature B.V. 2019

Abstract
Understanding patterns of speciation and subsequent gene flow can clarify the evolutionary origins and history of species
endemic to a specific geographic region and reveal genetic patterns important for conservation and management of rare
species. We chose Dicerandra from the North American Coastal Plain biodiversity hotspot as a model to explore these con-
cepts because of its endemism and the threatened status of most of its species. Using microsatellite-based population-level
analyses of 32 populations from four of the annual species (D. linearifolia var. linearifolia, D. linearifolia var. robustior,
D. fumella, D. odoratissima, and D. radfordiana), we addressed questions of genetic diversity, population structure, and
hybridization. Strong support was found for the species-level recognition of the recently described D. fumella from the
Florida panhandle. Dicerandra linearifolia var. linearifolia showed some regional cohesion of populations, but there was
no consistent geographic pattern to the clustering of populations. Dicerandra radfordiana showed consistent clustering with
proximate populations of D. odoratissima. Given that D. radfordiana is found at the southeastern extreme of the range of D.
odoratissima, these populations may represent the early stages of speciation by isolation. While there are morphological and
bioclimatic niche distinctions between D. odoratissima and D. radfordiana, there is no molecular support for a distinct D.
radfordiana. Overall, there is modest genetic diversity found at the population level for all Dicerandra annuals. Microsatellite
data support previously proposed hypotheses of hybridization between D. linearifolia var. linearifolia and D. odoratissima,
but do not support such hypotheses for D. fumella and D. linearifolia var. robustior.

Keywords Genetic structure · Population isolation · Endemism · Microsatellites · Dicerandra · Conservation

Introduction

Endemic organisms are valuable biologic and intrinsic com-

Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10592-019-01154-8) contains
supplementary material, which is available to authorized users.
* Adam C. Payton
acpayton@ufl.edu
* Andre A. Naranjo
aanaranjo@ufl.edu
1
Florida Museum of Natural History, University of Florida,
PO Box 117800, Gainesville, FL 32611-7800, USA
2
Department of Biology, University of Florida, PO
Box 118525, Gainesville, FL 32611-8525, USA
ponents of ecosystems. Analyses of endemics can provide
insights into the recent evolutionary history of a region,
as many endemics are the result of recent speciation and
thus represent the leading edge of the evolutionary process
(Givnish et al 2008; Baldwin 2007). In contrast, endemics
may be among the last remaining members of ancient com-
munities and can offer a glimpse into the past (Kruckeberg
and Rabinowitz 1985). In addition to understanding the
evolutionary history of the species, studying the patterns of
genetic diversity in narrowly distributed species can reveal
the ways in which isolation, selection, and drift contribute to
speciation. Understanding how genetic diversity is distrib-
uted across a landscape thus provides complementary insight
to ecological studies and can form a valuable framework for

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Non-monophyly of a species can result from incomplete
lineage sorting or from gene flow via ancient or recent
hybridization. Disentangling these processes can be chal-
lenging (e.g., Edwards et al. 2008; Rieseberg and Soltis
1991; Wendel and Doyle 1998; Folk et al. 2018a, b; Blis-
chak et al. 2018; Degnan 2018) but identifying occurrences
of intraspecific non-monophyly in phylogenetic studies
presents opportunities for more in-depth genetic analyses
at the population level. The speciation process is generally
not instantaneous, but is inherently continuous and dynamic
(Petit and Excoffier 2009; Nosil 2008). Analyses of genetic
diversity, population structure, and hybridization are criti-
cal for investigation of evolutionary history, speciation, and
species boundaries (Wheeler and Meier 2000). Population-
level sampling with highly variable markers is the best way
to investigate these questions. This study aims to address
the phylogenetic discontinuities revealed by Oliveira et al.
(2007) utilizing highly variable microsatellite markers and
range-wide population-level sampling of individuals. We
specifically focused on D. linearifolia, D. odoratissima,
D. fumella, and D. radfordiana as these species showed
phylogenetic incongruence among populations, have been
hypothesized to hybridize, and/or have extremely limited
distributions. Dicerandra densiflora was not included at this
time due to its phylogenetic monophyly and lack of evidence
for hybridization with other species.
Goals
We used analyses of genetic diversity, population structure,
and hybridization to provide insights into the evolutionary
history and species boundaries in the annual clade of Dic-
erandra. We considered the implications for conservation
of these species with extensions to the North American
Coastal Plain as a whole. Analyses focused on the follow-
ing: (1) assessing the genetic structure of all species and
determining if structure corresponds to geographic distribu-
tion and proposed phylogenetic relationships; (2) evaluating
hypothesized hybrid zones in the Florida panhandle between
D. fumella and D. linearifolia var. robustior and in eastern
Georgia between D. linearifolia var. linearifolia and D. odo-
ratissima; and (3) evaluating the genetic divergence between
D. radfordiana, the only annual with an extremely limited
range, and D. odoratissima.
Materials and methods
Sampling
During 2010 and 2011, 47 populations of Dicerandra were
sampled from Florida, Georgia, and southern Alabama
(Fig. 2), with either 16 individuals (38 populations) or 8
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Conservation Genetics
individuals (9 populations) per population; 32 populations
were selected for molecular analysis. Sampling locations
were initially identified through herbarium collections,
with additional locations discovered through exploration
of similar habitat in surrounding areas. Voucher specimens
were collected at all sites and deposited at the University of
Florida herbarium (FLAS).
454 sequencing
Because no microsatellite resources existed for Diceran-
dra, we used random shotgun 454 sequencing to generate
long-read sequences for microsatellite discovery and primer
design using an individual of D. linearifolia var. robustior.
Library preparation and sequencing were performed at the
Interdisciplinary Center for Biotechnology Research (ICBR)
at the University of Florida on a Roche 454 GS-FLX (Roche
Diagnostics Corp., Indianapolis, IN, USA). Sequence data
were trimmed and filtered for quality using FASTX Toolkit
(Gordon 2008).
Microsatellite discovery and genotyping
Using the 454 sequencing reads, microsatellite discovery
and primer design were accomplished using a modified Perl
script originally published by Castoe et al. (2010) and modi-
fied by M. Gitzendanner (Clivati et al. 2012). This script
searched the sequence data identifying microsatellite regions
followed by primer searches in the flanking regions using
Primer 3 (Rozen and Skaletsky 2000). Microsatellite sites
were manually checked to ensure perfect repeats with only
short homopolymer runs in the flanking region.
Genotyping was performed via single-plex PCR with
fluorescently labeled primers (Schuelke 2000). Up to 4 PCR
products per individual were pooled and analyzed for frag-
ment length at ICBR. GeneMarker v.1.6 (SoftGenetics LLC,
State College, PA, USA) was used to visualize chromato-
grams and design automated allele calling panels. All allele
calls were verified manually.
Genetic diversity
Loci were tested for linkage disequilibrium (LD) and
Hardy–Weinberg equilibrium (HWE) using GENEPOP v.4.1
(Rousset 2008). Arlequin v.3.5.1.2 (Excoffier and Lischer
2010) was used to calculate observed and expected heterozy-
gosity. GenAlEx v.6.4 (Peakall and Smouse 2006) generated
descriptive statistics, and F-statistics were calculated with
GENEPOP v.4.1. Pairwise comparisons of FST and Dest for
all populations were calculated using Arlequin v3.5.1.2 and
GENODIVE v.2.0 (Meirmans and Van Tienderen 2004),
respectively.
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