Emerging trends in
molecular systematics and molecular phylogeny of mayflies (Insecta:
Ephemeroptera)
K.G.
Sivaramakrishnan 1, K.A. Subramanian 2, M. Arunachalam3, C. Selva Kumar 4 & S. Sundar 5
1 Flat No.3,
Rams Apartments, No.7 Natesan Street, T. Nagar, Chennai, Tamil Nadu 600017,
India
2 Zoological
Survey of India, Western Regional Station, P.C.N.T. Post, Rawet Road, Akurdi,
Pune, Maharastra 411044, India
3,4,5 Sri Paramakalyani
Centre for Environmental Sciences, Manonmaniam Sundaranar University,
Alwarkurichi, Tamil Nadu 627412, India
Email: 1kgskrishnan@gmail.com, 2 subbuka.zsi@gmail.com (corresponding
author), 3 arunacm@gmail.com, 4 selvaaa06@gmail.com, 5 sundarstreco@gmail.com
Date of
publication (online): 26 August 2011
Date of
publication (print): 26 August 2011
ISSN
0974-7907 (online) | 0974-7893 (print)
Editor: V.V.
Ramamurthy
Manuscript details:
Ms # o2661
Received 29 December 2010
Final received 21 July 2011
Finally accepted 29 July 2011
Citation:Sivaramakrishnan, K.G., K.A. Subramanian, M. Arunachalam, C.S. Kumar & S.
Sundar (2011). Emerging trends in molecular systematics and molecular phylogeny
of mayflies (Insecta: Ephemeroptera). Journal
of Threatened Taxa 3(8): 1975–1980.
Copyright: © K.G.
Sivaramakrishnan, K.A. Subramanian, M. Arunachalam, C.
Selva Kumar & S. Sundar 2011. Creative Commons
Attribution 3.0 Unported License. JoTT allows unrestricted use of this
article in any medium for non-profit purposes, reproduction and distribution by
providing adequate credit to the authors and the source of publication.
Author Details: Dr. K.G. Sivaramakrishnan has been
working on aquatic insects especially on the systematics and biogeography of
mayflies (Ephemeroptera) of India over last 35 years. He has many publications
on the subject in international and national journals. Currently he is based in
Chennai.
Dr. K.A. Subramanian is a
scientist at Zoological Survey of India, Kolkata and has been working on
aquatic insects since 1998.
Dr. M. Arunachalam Professor at
Sri Paramakalayni Centre for Environmental Sciences, Manonmaniam Sundaranar
University, Alwarkuruchi. He is a freshwater biologist and
Ichthyologist and has been working on ecology and systematics of freshwater
organisms, especially the freshwater fishes.
C. Selva Kumar Doctoral
student at Sri Paramakalayni Centre for Environmental Sciences, Manonmaniam
Sundaranar University, Alwarkuruchi. Currently working on Ephemeroptera of
Kalakkad-Mundanthurai Tiger Reserve.
S. Sundar Doctoral student
at Sri Paramakalayni Centre for Environmental Sciences, Manonmaniam Sundaranar
University, Alwarkuruchi. Currently working on Naucoridae (Hemiptera) of
southern Western Ghats.
Author Contribution: KGS and KAS
conceived and prepared the review. MA actively participated in the discussion
of preparation of the manuscript and provided critical inputs. CSK and SS
helped in compiling the literature.
Acknowledgements: K.G.Sivaramakrishnan is grateful to Dr. M. Arunachalam for
having invited him to Sri Paramakalyani Centre for Environmental Sciences,
Manonmaniam Sundaranar University, Alwarkurichi, Tamilnadu,
India. He is indebted to authorities of Manonmaniam Sundaranar University for
having offered facilities to carry out a UGC Major Project, during which period
he could interact with co-authors to organize this review. K.A.Subramanian is
grateful to the Director, Zoological Survey of India for providing facilities
to prepare the manuscript.
Abstract: Current trends are reviewed in the molecular systematics and phylogeny
of the Ephemeroptera (mayflies), an ancient monophyletic lineage of pterygote
insects. Theories of mayfly
origins are analyzed, followed by a discussion of higher classification schemes
in light of recent developments in molecular systematics. Ephemeroptera evolution is a classic
example of ancient rapid radiation, presenting challenges for phylogenetic
analysis. The utility of combined
studies of morphological and molecular data is substantiated with examples and
the role of molecular systematics in unraveling the taxonomy of cryptic species
complexes is highlighted. The
importance of DNA barcoding in mayfly taxonomy is discussed in the light of
recent progress, and future contributions of genetics to the study of taxonomy,
ecology and evolution in mayflies are discussed.
Keywords: Cryptic species, DNA barcoding, Ephemeroptera, molecular phylogeny,
molecular systematics.
INTRODUCTION
The
order Ephemeroptera presently encompasses over 3000 species and 400 genera,
constituting at least 42 described families (Barber-James et al. 2008). The Ephemeroptera (mayflies) are an
archaic lineage of insects, dating back to the late Carboniferous or early
Permian periods, some 290 mya (Brittain & Sartori 2003). They occupy freshwater and brackish
water habitats across the world, with the exception of Antarctica. The nymphs are immature stages
inhabiting lentic and lotic waters. The imagos or adults are terrestrial; they lack mouth
parts and do not feed, relying on nutritional build up during immature
stages. They have an ephemeral
lifespan of a day or two and their only function is reproduction. The presence of a subimago with functional
wings at the penultimate moult is unique to pterygote insects. The winged stages of Ephemeroptera, as
with Odonata (dragonflies and damselflies) and the extinct Palaeodictyoptera,
cannot fold their wings horizontally over the abdomen as neopterans can.
This
article briefly reviews current trends in the molecular systematics and
phylogeny of the Ephemeroptera and discusses how combined analysis of
morphological and molecular data can be used to fine tune phylogenetic
conclusions.
Mayfly origins
The phylogenetic position of Ephemeroptera
within the winged insects (Pterygota) is hotly debated by systematists,
and significant disagreement still exists in morphological and molecular
studies. The
first complete mitochondrial genome of a heptageniid mayfly, Parafronurus youiwas sequenced using a long PCR-based approach by Zhang et al.(2008). In their analysis, the
basal Ephemeroptera hypothesis (Ephemeroptera versus (Odonata + Neoptera)) was
supported. This result also
received strong support by the nucleotide and amino acid datasets from
mitochondrial protein-coding genes with BI and ML analyses. Zhang et al. (2008)
tentatively concluded that mitochondrial genomes cananswer the difficult question of the basic relationships among the winged
insects. Ephemeroptera evolution
is a classic example of “ancient rapid radiation of insects” presenting
challenges for phylogenetic analysis because such radiations take place over
short periods of time and allow few distinctive phylogenetic markers to
accumulate among lineages.
The
Ephemeroptera, Odonata and Neoptera present a challenging phylogenetic tree
shape, regardless of their true relationships, because the first pterygotes may
have emerged up to 400 mya, but the earliest representatives of their extant descendants
is much younger than the first emergence of the lineage whose relationships are
in question (Whitfield & Kjer 2008).
Molecular systematics and higher classification
The
original subordinal classification of McCafferty & Edmunds (1979), based
mostly on thoracic morphology and wing pad position, comprised the holophyletic
suborder Pannota and the paraphyletic suborder Schistonota indicating the
retention of certain plesiomorphic (ancestral) traits. It was realized that monophyly derived
from synapomorphy (shared derived characters) should be the driving force
behind any taxonomic classification (Hennig 1966, 1979; Farris 1979). Later, McCafferty (1991) proposed 3
different suborders (Pisciforma, Setisura and Rectracheata) and traced
phylogenetic relationships within and among the suborders. Concurrent to McCafferty’s work, Kluge
(1988, 1998) independently proposed two suborders for Ephemeroptera. His suborder Furcatergalia is
equivalent to McCafferty’s Rectracheata, except the exclusion of Oniscigastridae
form Furcatergalia. The other
suborder proposal (Kluge 1988) was Costatergalia, which is equal to McCafferty’s
(1991) Pisciforma + Setisura + Oniscigastridae. Topological comparison of Kluge’s system and McCafferty’
system of mayfly classification is presented in Fig. 1, after Ogden et al.
(2009). In contrast to previous
hypotheses based on morphological observations, the relationships inferred from
the molecular data (Ogden & Whiting 2005) were congruent in some cases, but
incongruent in others. In their
analysis, the groups, Furcatergalia, Pannota, Carapacea, Ephemerelloidea and
Caenoidea and 15 families were supported as monophyletic. On the other hand, Setisura,
Pisciforma, Baetoidea, Siphlonuroidea, Ephemeroidea, Heptagenoidea and five
families (having more than one taxon represented) were not supported as
monophyletic.
However,
evidence supports the notion that combined data (morphology + molecular data)
analysis provides a more robust estimate of phylogenetic relationships. The study of Ogden et al. (2009)
represents the first formal morphological and combined (morphological and
molecular) phylogenetic analyses of the order Ephemeroptera. Taxonomic sampling comprised 112
species in 107 genera, including 42 recognized families (all major lineages of
Ephemeroptera). Morphological data
consisted of 101 morphological characters. Molecular data were acquired from DNA sequences of 12S, 16S,
18S, 28S and H3 genes. The Asian
genus Siphluriscus(Siphluriscidae) was supported as sister to all other mayflies. The lineages Carapacea, Furcatergalia,
Fossoriae, Pannota, Caenoidea and Ephemerelloidea were supported as
monophyletic. However, some
recognized families (for example, Ameletopsidae and Coloburiscidae) and major
lineages (such as Setisura, Pisciforma and Ephemeroidea among others) were not
supported as monophyletic, mainly due to convergences within nymphal characters
(Ogden et al. 2009).
Efficacy of combined morphological and molecular
phylogeny and systematics - examples from the Ephemeroptera
It
is quite obvious that most previous reconstructions of phylogeny and
classification were strongly hampered by superficial external morphological
similarities, which do not always reflect the true phylogeny of the Order. Homoplasies therefore seem a dominant
trait in mayfly morphology and behaviour, especially in nymphs (Ogden et al.
2009) and combined analysis may solve many riddles. Apart from the outstanding recent contribution of Ogden et
al. (2009) on these lines towards evolving a new paradigm in mayfly phylogeny,
some families notably, Leptophlebiidae (O’Donnell & Jockusch 2008),
Baetidae (Gattolliat et al. 2008) and Ephemerellidae (Ogden et al. 2009) have
received considerable attention regarding intrafamilial relationships, in which
molecular phylogenetic tools were extensively employed.
Using
two nuclear genes (the D2 + D3 region of 28S ribosomal DNA and histone H3) and
maximum parsimony (MP), maximum likehood (ML) and Bayesian inference (BI), O’Donnell
and Jockusch (2008) inferred the evolutionary relationships of 69
leptophlebiids sampled from six continents and representing 30 genera plus 11
taxa of uncertain taxonomic rank from Madagascar and Papua New Guinea. Although they did not recover monophyly
of the Leptophlebiidae, monophyly of two of the three
leptophlebiid subfamilies, Habrophlebiinae and Leptophlebiinae, was
recovered with moderate to strong support in most analyses. The Atalophlebiinae was rendered
paraphyletic as a result of the inclusion of numbers of Ephemerellidae or the
Leptophlebiinae clade. For the
species-rich Atalophlebiinae, four groups of taxa were recovered with moderate
to strong branch support: (i) an endemic Malagasy clade, (ii) a Paleoaustral
group, a pan-continental cluster with members drawn from across the Southern
Hemisphere, (iii) the Choroterpesgroup uniting fauna from North America, southeast Asia and Madagascar and (iv)
a group uniting three new world genera, Thraulodes, Farrodes and Traverella.
Gattolliat
et al. (2008) reconstructed the first comprehensive molecular phylogeny of the
Afrotropical Baetidae. They
sequenced a total of ca. 2300 bp from nuclear (18S) and mitochondrial (12S and
16S) gene regions from 65 species belonging to 26 genera. They used three different approaches of
phylogeny reconstruction viz., direct optimization, maximum parsimony and
maximum likelihood. The molecular
reconstruction indicated the Afrotropical Baetidae require a global revision at
a generic as well as suprageneric level.
The
investigation of Ogden et al.(2009) represented the combined molecular and morphological analysis for the
mayfly family Ephemerellidae (Ephemeroptera), with a focus on the relationships
of genera and species groups of the subfamily Ephemerellinae. The phylogeny was
constructed based on DNA sequence data from three nuclear (18S rDNA, 28S rDNA,
histone H3) and two mitochondrial (12S rDNA, 16S rDNA) genes, and 23
morphological characters. Ephemerella,the largest genus of Ephemerellidae, and Serratella were not supported as monophyletic
lineages. Strongly supported as
monophyletic include a grouping of the Timpanoginae genera Timpanoga, Dannella, Dentatellaand Eurylophella,
and groupings of the Ephemerellinae genera Torleya, Hyrtanella and Crinitella and the genera Kangella, Uracanthella and Teloganopsis. Further study and analysis of Ephemerellidae morphology is needed, and
classification should be revised, if it is to reflect true phylogenetic
relationships (Ogden et al. 2009).
Molecular systematics and cryptic species complex
Genetic
studies have highlighted cryptic diversity in many well-known taxa including
aquatic insects, with the general implication that there are more species than
are currently recognized. Baetis rhodani Pictet are among the
most widespread, abundant and ecologically important of all European mayflies
(Ephemeroptera), and used widely as biological indicators of stream
quality. Traditional taxonomy and
systematics have never fully resolved differences among suspected cryptic
species in the B. rhodanicomplex because morphological characters alone do not allow reliable
distinction. This is particularly true among larvae, the life-stage used most
widely in biomonitoring studies. Williams et al. (2006) assessed the molecular diversity of this complex
in one of the largest such studies of cryptic species in the order
Ephemeroptera to date. Phylogenies
were constructed using data from the mitochondrial cytochrome oxidase submit I
(COI) gene. Two monophyletic
groups were recovered consisting of one major haplogroup and a second clade of
six smaller but distinct haplogroups. Haplogroup divergence ranged from 0.2–3
% (within) to 8–19 % (among) with the latter values surpassing maxima
typically reported for other insects, and provided strong evidence for cryptic
species in the B. rhodanicomplex. However, the taxonomic
status of these seven haplogroups remains to be defined clearly.
The
potential implications of cryptic species in the B. rhodani complex on current and future ecological
studies are particularly far-reaching given the large number of studies carried
out on what now appears to be several possible distinct taxa. These results have wider relevance
since cryptic species have been detected in other aquatic insects (Jackson
& Resh 1998), and the presence and diversity of several taxa are widely
used as biological indicators (Mason 1996). The presence of cryptic species also has ramifications for
the assessment of biodiversity in general, and the ability to account for them
in future studies emphasizes the need to correlate genetic differences from
multi-locus data, with identifiable morphological characters and/or other
factors including physiology.
Dna barcoding and mayfly taxonomy
The
tool of DNA barcoding shows great potential for use by those studying the
systematics of many Ephemeroptera species groups. One example of the utility of barcoding is the verification
of stage associations, especially those not made by careful rearing. Recent revisionary study, on the family
Ephemerellidae Klapalek provides an illustration. The species concept of Ephemerella altana Allen, a western Nearctic taxon, had been
based on a larva belonging to the genus Ephemerella Walsh and an adult of Serretella Edmunds. Had barcoding technology been available at the time of E. altana’s discovery and description, it potentially
could have shown that this association was erroneous. Furthermore, barcoding could have helped to resolve the
species identities of the larva and adult. Based on traditional specimen
comparisons, the larva is thought to be that of the transcontinental species, Ephemerella excrucians Walsh, and the adult to
be that of the western species, Serretella
micheneri (Traver). Ephemerella excrucians exhibits an amazing
amount of morphological variability throughout its wide geographic range, which
begs the question of whether the current species concept might contain various
cryptic lineages that are unrecognizable by traditional, morphological
means. Barcoding technology could
be used to study various populations, including those from type localities, and
could provide a guideline for decisions about species identities and boundaries
(Zhou et al. 2008). Zhou et al.
(2009) have made a pioneering attempt to generate a DNA barcode reference
library for three insect orders- Ephemeroptera, Plecoptera and Trichoptera at
one site in the Canadian subarctic. This study has demonstrated that DNA barcoding holds great promise as a
tool for rapid biodiversity assessment in unknown faunas. A very close correspondence was
observed between morphospecies as determined by taxonomic experts and barcode
clusters designated using a standard sequence threshold. Several cases of proposed splitting may
reflect cryptic species.
DNA
barcodes of stream mayflies will improve descriptions of community structure
and water quality for both ecological and bioassessment purposes (Sweeney et
al. 2011). Rapid assessment of
biodiversity will aid the selection of sites of special conservation value and
will help to focus the efforts of taxonomists in revising and characterizing
the diversity of life (Zhou et al. 2009).
CONCLUSIONS
Future
perspectives on systematics and phylogeny of Ephemeroptera using recent
molecular tools are highlighted below:
-
Four sequencing markers which are well-surveyed and informative across a range
of divergences viz., the mitochondrial COI and 16S genes, and the nuclear 18S
and EF-Iα genes are suggested as standards for comparison for insect
molecular systematic studies (Caterino et al. 2000).
-
Species delineation continues to be one of the primary applications of genetic
techniques. Application of the Generalized Mixed Yule-coalescent (GMYC) model
to species circumscription using single-locus DNA appears rewarding (Pons et
al. 2006; Fontaneto et al. 2007). This approach has been applied successfully to 64 species of mayflies in
Madagascar (Monaghan et al. 2009).
-
Investigators of the demographic history of closely related populations or
species can use several nuclear DNA sequences to test specific hypotheses of
how past geological events influence
observed patterns (e.g. Knowles et al. 2007). These techniques allow one to test a priori hypotheses rather than post hoc
conclusions from patterns (Monaghan & Sartori 2009).
-
Wilcock et al. (2005) demonstrated very well how the combination of ecological
and genetic research, applied to several parts of the life cycle, can greatly advance our understanding of how populations
function in nature.
-
Routine sampling of population- and species- level genetic diversity, combined
with coalescent-based methods of species delineation has great potential to
become a standard procedure for the study of poorly known taxonomic groups like
Ephemeroptera (Gattolliat & Monaghan 2010).
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