Complex evolutionary history of the American Rubyspot damselfly, Hetaerina americana (Odonata): Evidence of cryptic speciation
Graphical abstract
Introduction
The current distribution of species is a result of several historical and contemporary factors, including dispersal, vicariance and gene flow (Avise, 2000). Analyzing how the distribution of evolutionary lineages changes over time allows us to test hypotheses about historical demographic size changes, secondary contacts, refugia and speciation patterns (Futuyma and Kirkpatrick, 2017). In odonates, understanding the factors that have shaped species distribution and genetic structure may be difficult because of their complex life cycle and behavior. For example, previous studies in this insect group have shown that some species are constituted by a single panmictic population at a global scale (Troast et al., 2016), while others show isolation by distance within few kilometers (Watts et al., 2004). These differences seem to be related to the dispersal capacity of the individuals of each species as well as to their territorial or migratory behavior. Also, the effects of particular physical barriers and the Quaternary glacial cycles have also been documented for some species (Callahan and McPeek, 2016, Jones and Jordan, 2015).
Genetic divergence can also be related to the evolution of reproductive isolation barriers. In odonates, the most common pre-zygotic barriers are sexual and mechanical or sensory isolation (Battin, 1993, Sánchez-Guillén et al., 2014a, Sánchez-Guillén et al., 2014b, Svensson and Waller, 2013). Sexual isolation has been associated with the coloration of individuals, such as in Calopteryx (Svensson et al., 2016, Tynkkynen et al., 2008), Mnais (Hayashi et al., 2005) and Argia (Nava-Bolaños et al., 2016), in which differences in wing and body coloration result in assortative mating, leading to genetic differentiation even among populations within species (Svensson et al., 2004). In turn, mechanical or sensory isolation is mainly related to the morphological configuration of clasping structures in males and the prothorax or head in females. The clasping structures (i.e. the caudal appendages) are structures found at the end of the abdomen of the males and used to grasp females and form the tandem position during copulation (Corbet, 1962). It has been suggested that besides the mechanical compatibility, there must be tactile stimuli for the female to accept to copulate (Barnard et al., 2017). The shape of the caudal appendages is well known to be species-specific in several genera of zygoptera (Barnard et al., 2017, Sánchez-Guillén et al., 2014b), but the role of intraspecific variation of these structures on mating patterns, genetic differentiation and incipient speciation processes has been barely analyzed (Barnard et al., 2017).
Despite that existence of more than 5000 species of odonates, to date the analyses of genetic differentiation and phylogeographic patterns are limited to a few species, mostly Palearctic and Nearctic species (Ferreira et al., 2016, Kohli et al., 2018, Swaegers et al., 2014; but see Feindt et al., 2014, Fincke et al., 2018, Sánchez-Herrera et al., 2010). In this study, we focused on Hetaerina americana (Calopterygidae), a broadly distributed species with a range from southeastern Canada to Nicaragua. Species of Hetaerina (commonly known as rubyspots), are characterized by the presence of a red spot at the base of the wings of male individuals and a territorial behavior. Most rubyspots are found along the neotropical region, with the highest species richness being found in South America (Garrison, 1990). Species identification within this genus has been problematic since many of them are very similar in wing and body coloration, body size, and are usually sympatric. Therefore, the main traits that allows an unambiguous species recognition are the morphology of the male caudal appendages (Garrison, 1990).
Hetaerina americana has been reported to occur in a variety of habitats, from temperate to tropical forests, and frequently is the most abundant species when it is in sympatry with other rubyspot species. Hetaerina americana is morphologically variable in the shape of the caudal appendages of the males and other traits such as the presence or absence of the pterostigma (i.e. a specialized colored cell in the outer wings, related to gliding control during flight) and the relative size of the red spots in the wings. Due to this variability, several synonymous taxa have been described (for example, H. pseudoamericana, H. texana, H. californica, etc.) (Garrison, 1990).
Hetaerina americana has been widely used as a model system in behavioral studies due to its complex mating system (Contreras-Garduño et al., 2008, Grether, 1996, Raihani et al., 2008). Males are territorial and display a lekking behavior (Córdoba-Aguilar et al., 2009), in which groups of males defend territories in the riverbanks and perform flying displays while females visit these areas to mate and do not receive other resources from males (nuptial gifts, oviposition sites). However, the population genetic structure of this species has not been studied and therefore no information is available regarding gene flow patterns and population history throughout its range. The only previous study was performed by Vega-Sánchez (2013), in which three widely separated populations of the species were analyzed using nuclear DNA sequences of the internal transcribed spacer 1, the 5.8S ribosomal RNA gene and the transcribed spacer internal 2 (ITS1-5.8S-ITS2) region. The populations analyzed were from Colorado in USA, and from Veracruz and Oaxaca in Mexico. The results showed a very high genetic differentiation among the three populations (ΦST > 0.60) and, remarkably, the presence of two similarly highly differentiated sets of individuals within the Veracruz population (Vega-Sánchez, 2013). Even though geographic isolation may partially explain these results, the considerable genetic differentiation among individuals within the Veracruz population indicates that other mechanisms of genetic isolation may be operating at a local level and suggesting that H. americana may be a complex of cryptic species. To test this hypothesis, in this study we performed a detailed phylogeographic and genetic structure analysis of H. americana throughout its distribution range using sequences of a fragment of the mitochondrial gene cytochrome oxidase I (COI) and the nuclear ITS1-5.8S-ITS2 region, as well as six nuclear microsatellites. Additionally, we employed geometric morphometric techniques to analyze and compare the shape variation of the male caudal appendages. The specific questions addressed were (i) what are the patterns of population genetic structure in H. americana throughout its distribution range? (ii) is the morphological variation of the male caudal appendages congruent with genetic differentiation patterns? (iii) does the evidence support the cryptic speciation hypothesis in H. americana?
Section snippets
Sampling
Two hundred and twenty adult individuals of H. americana were directly collected or obtained through donations, representing a total of 31 localities (Supplementary Table 1), from Guatemala to the United States, covering most of its distribution range.
DNA extraction, amplification, sequencing and genotyping
Genomic DNA was isolated from thoracic muscle with the Pure Link Genomic DNA Mini Kit (Invitrogen®) following the protocol of the manufacturer. A fragment of the cytochrome oxidase I (COI; 658 pb) gene was amplified using the ODO_HCO2198d and
Mitochondrial genetic diversity and neutrality tests
Two-hundred and sixteen sequences of the COI region were obtained and deposited at GenBank (see Supplementary Table 1 for accession numbers). In total, 61 haplotypes were observed with high haplotypic and nucleotide diversity (see Supplementary Table 3). The haplotype network based on statistical parsimony showed four major haplogroups (Fig. 1A). The first one (in green) included haplotype H9, which had the highest frequency (81 individuals), and other 26 haplotypes, which are singletons or
Discussion
The results of this study show complex patterns of genetic and morphological variation within H. americana. Levels of genetic differentiation were very high for the three sets of molecular markers used (mitochondrial COI and nuclear ITS and microsatellites), but the COI variation was strongly discordant with the pattern shown by both nuclear markers. On the other hand, morphological variation in the male caudal appendages was completely congruent with nuclear genetic variation. Taken together,
Acknowledgements
We thank Eliot Camacho, Ricardo Durán, Mariana Solís, Luis Letelier, Aly Valderrama, Sergio Marcos, Gonzalo Contreras, Elsa Hernández, Hernando Rodríguez and Tamara Ochoa for helping in field collection. We especially thank George Sims for sending us samples from USA. Jesús Llanderal and Víctor Rocha provided technical help with microsatellites sequencing and Orlando Hernández for the microscopy photographs of caudal appendages. Y. M. Vega-Sánchez thanks CONACyT (CVU 549239) and the Posgrado en
Funding source
This work was supported by the grant number MICH-2012-C05-197824, Fondos Mixtos CONACYT-Gobierno del Estado de Michoacán.
References (76)
- et al.
Cryptic species as a window on diversity and conservation
Trends Ecol. Evol.
(2007) - et al.
Multi-locus phylogeny and divergence time estimates of Enallagma damselflies (Odonata: Coenagrionidae)
Mol. Phyl. Evol.
(2016) - et al.
The ecology of nonecological speciation and nonadaptive radiations
TREE
(2019) - et al.
Male mating tactics in the American rubyspot damselfly: territoriality, nonterritoriality and switching behaviour
Anim. Behav.
(2008) - et al.
Adaptive radiation, nonadaptive radiation, ecological speciation and nonecological speciation
TREE
(2009) - et al.
A neotropical polymorphic damselfly shows poor congruence between genetic and traditional morphological characters in Odonata
Mol. Phyl. Evol.
(2010) - et al.
Finding evolutionary processes hidden in cryptic species
Trends Ecol. Evol.
(2018) - et al.
Hybridization in Calopteryx damselflies: the role of males
Anim. Behav.
(2008) - et al.
Phylogenetic relationships inferred from ribosomal its sequences and biogeographic patterns in representatives of the genus Calopteryx (Insecta: Odonata) of the West Mediterranean and adjacent West European zone
Mol. Phyl. Evol.
(2001) - et al.
Characterization of novel microsatellite loci for Hetaerina americana damselflies, and cross-amplification in other species
Conserv. Genet. Resour.
(2013)
Phylogeography: The History and Formation of Species
Mechanical and tactile incompatibilities cause reproductive isolation between two young damselfly species
Evol.
The odonate mating system, communication, and sexual selection: a review
Bolletino di zoologia
Morphometric Tools for Landmark Data: Geometry and Biology
BEAST 2: A software platform for Bayesian evolutionary analysis
PLoS Comput. Biol.
Experimental support that natural selection has shaped the latitudinal distribution of mitochondrial haplotypes in australian Drosophila melanogaster
Mol. Biol. Evol.
TCS: A computer program to estimate gene genealogies
Mol. Ecol.
The size of the red wing spot of the American rubyspot as a heightened condition-dependent ornament
Behav. Ecol.
A Biology of Dragonflies
The lek mating system of Hetaerina damselflies (Insecta: Calopterygidae)
Behaviour
JModelTest 2: More models, new heuristics and parallel computing
Nat. Methods
Redefining the damselfly families: a comprehensive molecular phylogeny of Zygoptera (Odonata)
Syst. Entomol.
BEAST: Bayesian evolutionary analysis by sampling trees
BMC Evol. Biol.
Phylogenetic relationships, divergence time estimation, and global biogeographic patterns of calopterygoid damselflies (Odonata, Zygoptera) inferred from ribosomal DNA sequences
Syst. Biol.
A molecular phylogeny of the Odonata (Insecta)
Syst. Entomol.
STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method
Conserv. Genet. Resour.
Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study
Mol. Ecol.
Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows
Mol. Ecol. Resour.
Still a one species genus? Strong genetic diversification in the world's largest living odonate, the Neotropical damselfly Megaloprepus caerulatus
Conserv. Genet.
Genetic distinctiveness of the damselfly Coenagrion puella in North Africa: an overlooked and endangered taxon
Conserv. Genet.
Worldclim 2: new 1-km spatial resolution climate surfaces for global land areas
Int. J. of Climatol.
Tests of hypotheses for morphological and genetic divergence in Megaloprepus damselflies across Neotropical forests
Biol. J. Linn. Soc.
Cryptic species as a window into the paradigm shift of the species concept
Mol. Ecol.
Nonshivering thermogenesis capacity associated to mitochondrial DNA haplotypes and gender in the greater white-toothed shrew, Crocidura russula
Mol. Ecol.
Evolution
A synopsis of the genus Hetaerina with descriptions of four new species (Odonata: Calopterygidae)
Trans. Am. Entomol. Soc.
Sexual selection and survival selection on wing coloration and body size in the Rubyspot damselfly Hetaerina americana
Evol.
PAST: paleontological statistics software package for education and data analysis
Palaeontologia Electronica
Cited by (11)
Evolutionary history and divergence times of Odonata (dragonflies and damselflies) revealed through transcriptomics
2021, iScienceCitation Excerpt :Phylogenetic relationships in Zygoptera have previously been considered chaotic, and a fully resolved phylogeny of Zygoptera does not yet exist. Past studies have only resolved parts of the Zygoptera family tree (Dumont et al., 2005; Sánchez-Herrera et al., 2020; Vega-Sánchez et al., 2019). Many of the traditionally recognized families, such as Amphipterygidae and Megapodagrionidae, as well as genera such as Agriocnemis and Pseudagrion have been shown to be polyphyletic (Dijkstra et al., 2014), which clearly hampers the compilation of a reliable taxon sampling to target deep phylogenetic relationships within Zygoptera.
A molecular phylogeny of forktail damselflies (genus Ischnura) reveals a dynamic macroevolutionary history of female colour polymorphisms
2021, Molecular Phylogenetics and EvolutionCitation Excerpt :In presenting these results, we hope to stimulate future empirical investigations on these and other damselfly clades, and we discuss some promising hypotheses for follow-up studies. We note that many damselfly groups, although popular in much ecological and evolutionary research (Cordoba-Aguilar, 2008) are still relatively poorly investigated with respect to phylogenetic relationships and evolutionary history, although some recent progress has been made, particularly within the family Coenagrionidae (Beatty et al., 2017; Callahan and McPeek, 2016; Dijkstra et al., 2014; Swaegers et al., 2014; Torres-Cambas et al., 2019; Vega-Sánchez et al., 2019). We have gathered DNA-sequence data, phenotypic data and geographic range size data for 41 species of Ischnura.
Niche differentiation, reproductive interference, and range expansion
2024, Ecology LettersReference genome for the American rubyspot damselfly, Hetaerina americana
2023, Journal of HeredityCourtship and mate-finding in insects: A comparative approach
2023, Courtship and Mate-finding in Insects: A Comparative Approach