Samantha Smith and Seth Bybee

Introduction:
Damselflies (Zygpotera) exhibit diverse mating behaviors, with the family Calopterygidae known for its pigmented wings and territorial behavior (Cordoba-Aguilar, Cordero-Rivera 2005). The genus Hetaerina, or rubyspot damselflies, has been used in numerous studies due to mating behaviors, morphology and a high density along certain streams (Anderson 2010, 2011; Contreras-Garduno 2006; Cordoba-Aguilar 2009; Grether 1996, 1997; Raihani 2008; Regions 2011). Despite use in studies, relationships between species of Hetaerina remain unclear. Over the past ten years, the taxonomy of Hetaerina and their sister genus Mnesarete has been in flux with species being transferred between the two genera and into Ormenophlebia and Bryoplathanon (Garrison 1990, 2006). In 2005 Dumont went so far as to suggest Hetaerina should be its own family, Hetaerinidae. However, the basic taxonomy of the group needs to be refined before any family designation can be properly assessed.
A phylogeny of Hetaerina will also provide an excellent tool for future research to examine mating strategies more deeply (Grether 1996; Regions 2011). Though many Hetaerina exhibit the lek mating system (i.e. males guard a territory without any resource) others like H. rosea, exhibit the resource defense polygyny strategy and guard the substrate where females oviposit (Regions 2011). In addition, Mnesarete pudica has precopulatory behavior not seen in any Hetaerina species (Guillermo-Ferreira 2012). A phylogeny of Hetaerina and Mnesarete will allow researchers to trace these behaviors and determine how such variation evolved.
The wing pigmentation or ruby spot that Hetaerina is known for is linked to male success in acquiring and holding territories (Contreras 2006). The larger the ruby spot, the larger the fat reserve and the higher chance of success males will have when competing for territories. The size of the ruby spot varies between species: for example, H. americana males have coloration on only the basal portion of the wing and H. longipes males have coloration on both the basal and apex wing portions. Interestingly, only two Mnesarete have pigmentation on their wings, M. pudica and M. fulgida. The rest of Mnesarete have hyaline wings. It is unknown to what extent wing pigmentation influences male success within Mnesarete, and what could have driven the evolution of hyaline wings. As the first step in uncovering these questions, I will create a phylogeny of Hetaerina and Mnesarete. My phylogeny will focus on the hypotheses: 1) Hetaerina is not monophyletic. 2) Hyaline-winged Mnesarete evolved once. 3) Non-hyaline winged Mnesarete evolved once.

Materials and Methods:
Taxon Sampling:
To increase the accuracy of the phylogeny I sampled extensively across Hetaerina and Mnesarete, including 14 Mnesarete species, 30 Hetaerina species, two Ormenophlebia, and one Calopteryx for a total of 47 species and 206 specimens. Within Hetaerina, 80% of the species are included in the phylogeny. Legs of specimens (for DNA work) were donated by the Florida State Collection of Arthropods, as well as by collaborators at UCLA, Rutgers University and UFSCAR. Multiple specimens from each species at different geographic locations were included to test for accuracy of species designations. Specimens were kept in ethanol following collection.
DNA extraction, amplification and sequencing:
All DNA extraction, amplification and sequencing was done in Dr. Seth Bybee’s lab at Brigham Young University. Sequence data were generated for all taxa. Four genes were targeted for amplification (12S, ITS1/ITS2, ef1a, COI/COII), using primers modified for Hetaerina. Genes were amplified using standard polymerase chain reaction (PCR) techniques in thermocycler. Yield and potential contamination was monitored by gel electrophoresis. Sequencing was performed at BYU sequencing center.
Phylogenetic analyses:
Sequencing data was uploaded to Geneious (v6.1.8), where it was edited and aligned. All genes were aligned using MAFFT in Geneious. Following alignment genes were also concatenated in Geneious. A maximum likelihood tree was created in IQtree. FigTree (v1.4.2) was used to view and analyze the phylogeny.

Results:
I recovered a maximum likelihood tree using IQtree (fig. 1). Neither Hetaerina nor Mnesarete is monophyletic, with Mnesarete reconstructed as four different lineages within Hetaerina, and the genus Ormenophlebia nested within Hetaerina as well.
The combined Hetaerina, Mnesarete and Ormenophlebia genera form a monophyletic clade. Hetaerina americana, H. auripennis, H. aurora, H. capitalis, H. cruentata, M. guttifera, H. hebe, H. longipes, H. majuscula, H. miniata, H. moribunda, H. occisa, H. pilula, H. rosea, H. sempronia, H. titia, H. vulnerata, and H. westfalli form monophyletic species groups, indicating that these were correctly identified as species. In addition, Mnesarete aenea, M. astrape, M. cuprea, M. drepane, M. hauxwelli, M. metallica, M. pudica, M. williamsoni, and Ormenophlebia imperatrix also form monophyletic species groups.
Either due to misidentification of species, or due to incorrect determination (i.e. they are not true species) H. amazonica, H. caja, H. charca, H. sanguinea and M. fulgida are not monophyletic as species.
Seven new species were added to the phylogeny due to fieldwork and collaboration in Brazil, funded by the ORCA grant.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Discussion/Conclusion:
Neither Hetaerina nor Mnesarete is monophyletic. Further work should be done to combine these two genera into a new genus. More behavioral data is needed to determine how consistent behavioral differences are between Hetaerina and Mnesarete. Though Ormenophlebia adults are morphologically very different from Hetaerina and Mnesarete, it too appears to be nested within Hetaerina. Ormenophlebia naiads are almost identical to Hetaerina however, which may support Ormenophlebia staying within Hetaerina (Garrison 2006). Including morphology with our phylogeny will allow for a better analysis as to the placement of Ormenophlebia. Hyaline wings evolved four times within the Hetaerina/Mnesarete clade, whereas non-hyaline wings are seen in only in Mnesarete pudica and Mnesarete fuscibasis. However, as they are within Hetaerina, they likely evolved from a basal rubyspot wing pattern, rather than from hyaline wings seen in other Mnesarete.
Our phylogeny opens the door for future behavioral studies of Hetaerina, Mnesarete and Ormenophlebia. As more behavioral and morphological data is provided, we can trace mating behaviors across the phylogeny. We can then hypothesize how male to male competition and female choice impacts the evolution characters such as the ruby spot, and how this impacts the evolution of mating behaviors. Understanding how male to male competition and female choice impact mating behavior is essential in discovering how traits are passed on within a species. Many other organisms, especially within birds and insects, are sexually dimorphic. As Hetaerina and Mnesarete females remain cryptic, it is highly likely that mating behavior is driving their speciation. A switch from intrasexual selection (male to male competition) to intersexual selection (female choice), as potentially seen in Hetaerina and Mnesarete, could be a driver of speciation in other organisms as well. This study has shed light on potential drivers of change in mating behavior and speciation across the animal kingdom.

Scholarly Sources
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