Molecular phylogenetics of the mosquito tribe Aedini
John Soghigian, Marco Notarangelo, and Todd P. Livdahl. Clark University, Worcester MA
Background
For most of the twentieth century, Aedes was a cosmopolitan genus that grew to
over 900 species.
In 2000, Reinert1 divided the genus into two genera based on sexual anatomy,
Aedes and Ochlerotatus. Quantitative morphological analyses 2 suggested
paraphyly within Aedes and Ochlerotatus, leading to the additional elevation of
more than 70 subgenera to the genus level. These elevations were not wholly
accepted by the community.
The problem is well illustrated with Aedes japonicus, a major invasive disease
vector in North America and Europe, whose range continues to expand today.
This mosquito was known as Ochlerotatus japonicus, then Hulecoeteomyia
japonica, and then back to Aedes japonicus, all in a ten year period. During this
period, scholars published extensively using both Aedes and Ochlerotatus for
generic epithets and different journals had different standards.
More recent analyses by Wilkerson et al.3 did not find support for these
additional groups using alternative methods on the same data set, thus returning
Aedes to a single large genus, but once more on morphological data alone.
Figure 1, Left – Invasive disease vector Aedes
japonicus. This species has been renamed several
times in the last decade, making communication
about the organism difficult between scholars and
the public.
Figure 2, Right– Invasive disease vector Aedes
albopictus. Though renamed to Stegomyia albopicta,
this nomenclature change was never adopted by the
majority of scholars (see Wilkerson et al. for details)
Why molecular methods?
While morphology is extremely useful for differential identification, morphological
systematics is limited by several factors (see Hillis 1987) relative to molecular
systematics, chief among them the limited amount of data available to
morphological studies; the present study contains an alignment of more than four
thousand characters, compared to 334 for previously referenced morphological
studies.
Figure 3 – Two examples of mosquito sexual anatomy which can be used
to differentiate many “Aedes” from “Ochlerotatus” mosquitoes.
We have likely reached the limits of what morphology alone can tell us about the
evolutionary history of the Culicidae, as evidenced by the conflicting results of
Reinert and colleagues compared with Wilkerson et al.
Limited molecular phylogenies of Aedes mosquitoes exist; those that do
generally support Reinert’s conclusions, but contain relatively few taxa 4,5.
Figure 4 – The maximum likelihood tree from PhyML with aBayes
support values, which can be interpreted similarly to Bayesian
posterior probabilities. Here we only show our analysis for the
clade containing the Aedini. Arrows highlight species outside of
the genus Aedes that are resolving within the Aedes clade.
Results
Clade A contains all species in this analysis classically associated with Aedes.
Furthermore, we found strong support for the groups proposed in Reinert 2000
in Aedes (B) and Ochlerotatus (C). However, each clades also contained a
representative of another genus: Armigeres subalbatus (within B) and
Haemagogus (within C). Thus, the classic Aedes appear to be paraphyletic,
though Reinert 2000’s Aedes appears monophyletic in this analysis. We could
not resolve the placement of Haemagogus (D; support values <0.6), and thus
can not reject the monophyly of the Ochlerotatus group.
A GenBank-driven database approach
We utilized the R package megaptera to build a database of GenBank
sequences. megaptera downloads desired markers, aligns conspecific
sequences, and creates a single consequence sequence from the alignment for
each species.
Then come our excellent collaborators, who have provided the majority of the
specimens of the 70 species currently stored at Clark. We generate sequences
and add them to each alignment, then re-align using MAFFT in Geneious.
Where possible, we always favor sequences generated within our laboratory,
replacing megaptera generated sequences where possible.
We used PhyML to generate our phylogeny with aBayes branch support values
under the GTR nucleotide substitution model.
Figure 5 – An example of our alignment from the enolase gene. Different colors
indicate different bases. We used sequences from COI, 18S, 28S, enolase, and
arginine kinase.
Discussion and future directions
Paraphyly of the genus Aedes
Our analysis suggests that the classical and currently recognized genus of
Aedes is paraphyletic. Armigeres appears to share a more recent common
ancestor with mosquitoes such as Aedes aegypti than Aedes aegypti share with
the Ochlerotatus mosquitoes, such as Aedes triseriatus. As monophyly is the
cornerstone of any genus, this suggests that the current generic status of
classical Aedes should be re-evaluated.
We will continue to build on this analysis with additional genera where possible,
while utilizing additional support methods, such as bootstrapping implemented
in RAxML.
Future comparative studies
Our phylogeny has uses beyond resolving taxonomic naming issues. For
instance, we have found significant support for cophylogeny between both
Ascogregarina and Aedini hosts, and Amblyosporidiae and Culicidae hosts
(data not shown). It is our hope that this phylogeny might be useful in future
comparative efforts, and might allow for a greater understanding of host and
disease coevolution, as well.
Acknowledgments
This project was made possible by the generous donations of multiple
collaborators, such as Michael Hutchinson and Ary Faraji, without whom this
project would never have been possible.
This project was partially supported by the Northeastern Mosquito Control
Association, NIH grant 1R15AI092577-01A1, NSF grant award number
1311535, and Clark University.
References
1.Reinert, J. F. New classification for the composite genus Aedes (Diptera: Culicidae: Aedini), elevation of subgenus Ochlerotatus to generic rank,
reclassification of the other subgenera, and notes on certain subgenera and species. J. Am. Mosq. Control Assoc. 16, 175–188 (2000).
2.Reinert, J. F., Harbach, R. E. & Kitching, I. J. Phylogeny and classification of tribe Aedini (Diptera: Culicidae). Zool. J. Linn. Soc. 157, 700–794
(2009).
3.Wilkerson, R. C. et al. Making Mosquito Taxonomy Useful: A Stable Classification of Tribe Aedini that Balances Utility with Current Knowledge of
Evolutionary Relationships. PLoS ONE 10, e0133602 (2015).
4.Shepard, J. J., Andreadis, T. G. & Vossbrinck, C. R. Molecular Phylogeny and Evolutionary Relationships Among Mosquitoes (Diptera: Culicidae)
from the Northeastern United States Based on Small Subunit Ribosomal DNA (18S rDNA) Sequences. J. Med. Entomol. 43, 443–454 (2006).
5.Reidenbach, K. R. et al. Phylogenetic analysis and temporal diversification of mosquitoes (Diptera: Culicidae) based on nuclear genes and
morphology. BMC Evol. Biol. 9, 298 (2009).