aquatic insect conservation: a molecular genetic approach

TECHNICAL REVIEW

Aquatic insect conservation: a molecular genetic approach

K. G. Sivaramakrishnan • S. Janarthanan •

C. Selvakumar • M. Arumugam

Received: 21 September 2013 / Accepted: 17 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Ecosystem diversity, species richness and

genetic diversity are the three major facets of biodiversity

which deserve equal attention for conservation. However,

genetic diversity is significant in identification of unique

populations for conservation purpose. Aspects of DNA

barcoding, loss of genetic diversity in cryptic species and

examples of molecular phylogenetic and molecular phy-

logeographic studies on aquatic insects of headwaters from

major biogeographic realms are briefly reviewed in the

light of prioritization of taxa and habitats for conservation.

Special emphasis is laid on identification of evolutionary

significant units for effective conservation in the context of

global climate change. Current methodologies for identi-

fying potential loss of intraspecific genetic diversity are

also highlighted with suggestions on future research

priorities.

Keywords Genetic diversity � Conservation � DNA

barcoding � Molecular phylogeography � Aquatic insects �Global climate change

Introduction

Approaches for setting conservation priorities are becom-

ing a matter of concern as the accelerating and potentially

catastrophic loss of biological diversity, unlike other

environmental threats, is irreversible (Mittermeier et al.

1998). Some of the key drivers affecting the loss of bio-

diversity worldwide are habitat fragmentation, habitat loss,

overexploitation, land use changes, nitrogen deposition,

alien species invasions and more recently climate change

impacts (Beheregaray and Caccone 2007; Heino et al.

2009). In this context, while great attention is being paid on

conservation of mega fauna, especially keystone and flag-

ship mammalian fauna and charismatic avian fauna,

awareness for the need to conserve the macroinvertebrate

communities inhabiting the fragile freshwater habitats is

very minimal especially in the developing world. Aquatic

insects are, by far the most abundant and species-rich

organisms inhabiting the freshwaters, which constitute 3 %

of the world’s water occupying just 1.9 % of the land

surface. They are the vital components of the freshwater

ecosystems in terms of being essential fish food and in

structuring lentic and lotic communities providing unique

ecosystem services especially in natural nutrient cycling.

Presently, socioeconomic, legal and ecological factors are

being taken into account to a greater or lesser degree by

conservation biologists while including insect conservation

as part of an overall strategy regarding maintenance of rea-

sonable biotic integrity and habitat heterogeneity of the

entomofauna of lentic and lotic waters. The emerging trend,

however is to augment the study of the impact of global

climate change on genetic diversity within populations and

species of imperiled taxa for prioritization for conservation.

Pauls et al. (2013) define genetic diversity as all the genetic

variants within and among populations of evolutionary lin-

eages, where evolutionary lineages can represent recognized

morphospecies, morphologically cryptic species or other

evolutionary significant units (ESUs). Significant milestones

in the study of conservation of genetic diversity using aquatic

insects as model organisms are presented in Table 1.Recent

K. G. Sivaramakrishnan (&)

Department of Zoology, Madras Christian College

(Autonomous), Tambaram East, Chennai 600059, India

e-mail: [email protected]

S. Janarthanan � C. Selvakumar � M. Arumugam

Department of Zoology, University of Madras, Guindy Campus,

Chennai 600025, India

123

Conservation Genet Resour

DOI 10.1007/s12686-014-0250-4

Table 1 Significant milestones in the study of conservation of genetic diversity using aquatic insects as model organisms

Taxon Order Aspects of investigation Authors

Baetis alpines Ephemeroptera Genetic differentiation Monaghan et al. (2001)

Rhithrogena loyolaea, Baetis alpines

and Allogamus auricollis

Ephemeroptera and

Trichoptera

Population genetic structure Monaghan et al. (2002)

Baetis rhodani Ephemeroptera Microsatellite loci Williams et al. (2002)

Archichauliodes diversus and

Coloburiscus humeralis

Megaloptera and

Ephemeroptera

Population genetic structures Hogg et al. (2002)

Elporia barnardi Dipter Genetic population structure Wishart and Hughes (2002)

Megalagrion Odonata Phylogeographic patterns Jordan et al. (2005)

Baetidae Ephemeroptera Predominant biogeographic force in

Madagascar

Monaghan et al. (2005)

Ephemeroptera Ephemeroptera DNA barcoding Ball et al. (2005)

Drusus discolor Trichoptera Phylogeography Pauls et al. (2006)

Coenagrion mercurial Odonata Population structure Watts et al. (2006)

Metacnephia coloradensis Diptera Population genetic structure Finn and Adler (2006)

Drusus discolor and D. romanicus Trichoptera Comparing the population structure Pauls et al. (2007)

Ochthebius glaber Coleoptera Conservation genetics Abellan et al. (2007)

Fallceon quilleri Ephemeroptera Dispersal ability and genetic structure Zickovich and Bohonak (2007)

Baetis venus Ephemeroptera Cryptic diversity revealed by mtDNA

barcodes COI

Stahls and Savolainen (2008)

Rhyacophila aquitanica Trichoptera Population genetic structure Balint et al. (2008)

Rhyacophila pubescens Trichoptera Population genetic structure Engelhardt et al. (2008)

Cheumatopsyche sp., Tasimia palpata

and Bungona narilla

Trichoptera and

Ephemeroptera

Population genetic structure Hughes et al. (2008)

Leptophlebiidae Ephemeroptera Molecular phylogeny O’Donnell and Jockusch

(2008)

Arcynopteryx compacta Plecoptera Microsatellite markers Theissinger et al. (2009)

Hydropsyche tenuis and Drusus

discolor

Trichoptera Contrasting patterns of population

structure

Lehrian et al. (2009)

Nebrioporus ceresyi and Ochthebius

notabilis

Coleoptera Parallel habitat-driven differences in the

phylogeographical structure

Abellan et al. (2009)

Ephemeroptera, Plecoptera and

Trichoptera

Ephemeroptera,

Plecoptera and

Trichoptera

DNA barcode library Zhou et al. (2009)

Chaetopterygopsis maclachlani Trichoptera Genetic population structure Lehrian et al. (2010)


Trichoptera

Ephemeroptera,

Plecoptera and

Trichoptera

DNA barcoding Zhou et al. (2010)

Ameletus inopinatus Ephemeroptera Modeling range shifts and assessing

genetic diversity distribution

Taubmann et al. (2011)

Ephemeroptera and Trichoptera Ephemeroptera and

Trichoptera

Environmental barcoding Hajibabaei et al. (2011)


Trichoptera

Ephemeroptera,

Plecoptera and

Trichoptera

DNA barcode reference library Zhou et al. (2011)

Palingenia longicauda Ephemeroptera Effects of human-induced range loss—

unexpected genetic diversity

Balint et al. (2012)

Ephemeroptera Ephemeroptera DNA barcoding Webb et al. (2012)

Arcynopteryx dichroa Plecoptera Glacial survival and post-glacial

recolonization

Theissinger et al. (2013)

Heptageniidae Ephemeroptera Coalescent and phylogenetic analysis Vuataz et al. (2013)

Abedus herberti Hemiptera Population genetic structure Phillipsen and Lytle (2013)

Trichoptera Trichoptera DNA barcoding Ruiter et al. (2013)


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trends in genetic aspects of aquatic insects’ conservation are

highlighted below indicating future research priorities in this

field.

DNA barcoding and conservation

Unprecedented ‘biodiversity erosion’ mainly due to habitat

fragmentation, global warming, climate change and other

negative anthropogenic impacts as pointed above has

necessitated formulation of stringent conservation measures.

In this context, precise species delineation, understanding

intraspecific diversity and cryptic species complexes are

very much essential. DNA barcode is a short (about 650 bp)

fragment of the mitochondrial cytochrome c oxidase subunit

1 (COI) gene (Hebert et al. 2003). The COI gene has been

used effectively to identify many eukaryotic species,

including invertebrates such as springtails (Hogg and Hebert

2004), mayflies (Ball et al. 2005), and chironomids (Pfenn-

inger et al. 2007; Sinclair and Gresens 2008), where specific

life history stages, damaged specimens, or cryptic species

are difficult to identify using morphological traits (Elderkin

et al. 2012). DNA barcoding, in particular, can contribute to

conservation policy in two important ways: by speeding up

local biodiversity assessments to prioritize conservation

areas or evaluate the success of conservation actions, and by

providing information about evolutionary histories and

phylogenetic diversity (Francis et al. 2010; Krishnamurthy

and Francis 2012). DNA barcodes act as cost effective tools,

quick and objective inputs to prioritize conservation efforts

(Yao et al. 2009; Dentinger et al. 2010; Li and Dao 2011).

Webb et al. (2012) while establishing a DNA Barcode

Library for North American Ephemeroptera found that

nearly 20 % of the species included two or three very distinct

haplotype clusters likely representing species complexes,

rather than individual species. Thus traditional ‘‘species’’

measures of diversity grossly underestimated overall diver-

sity with important implications in conservation.

Loss of genetic diversity in cryptic species

Cryptic species are classified as a single nominal species

because they are morphologically indistinguishable though

genetically differentiated (Bickford et al. 2007). Cryptic

diversity encompasses the diversity of genetic variations

within described species and can only be explored using

molecular-genetic tools. Cryptic diversity has been high-

lighted in aquatic insects from the orders Odonata,

Hemiptera, Trichoptera, Diptera and Ephemeroptera

(Williams et al. 2006). Without proper estimation of

cryptic species complexes, the effects of Global Climate

Change (GCC) at the most fundamental level of biodiver-

sity—intraspecific genetic diversity—has remained

elusive.

Balint et al. (2011) showed that the use of morphospe-

cies-based assessments of GCC effects will result in

underestimation of the true scale of biodiversity loss. Their

studies on species distribution modeling and assessments of

mitochondrial DNA variability in nine species of montane

aquatic insects in Europe indicated that future range con-

tractions will be accompanied by severe loss of cryptic

evolutionary lineages and genetic diversity within these

lineages. These losses have greatly exceeded those at the

scale of morphospecies. Their results demonstrated that

intraspecific pattern of genetic diversity should be consid-

ered when estimating the effects of climate change on

biodiversity. This opportunity will pave the way for using

genetic diversity to make conservation strategy more effi-

cient so that conservation areas may be prioritized where

both a suitable habitat for the species and a high degree of

intraspecific genetic diversity can be preserved in future.

Cryptic species can contribute to defining patterns of

biodiversity at nested spatial scales that may be important

for freshwater conservation (Cook et al. 2008). Cryptic

species and cryptic lineages are more or less evenly dis-

tributed among major metazoan taxa including insects and

in several biogeographic regions (Pfenninger and Schwenk

2007). While discussing various methods that could

objectively prioritize conservation below species level,

Fraser and Bernatchez (2001) reviewed ESU concepts and

proposed a synthetic approach of ‘‘adaptive evolutionary

conservation’’ (AEC) providing a context-based framework

for delineating ESUs. They concluded that differing ESU

approaches are only tools in the AEC tool box. They need

not conflict with one another but can operate in a com-

plementary and adaptive fashion.

Ecosystem diversity, species richness and genetic

diversity are the three major facets of biodiversity which

deserve equal attention for conservation. However, genetic

Table 1 continued

Taxon Order Aspects of investigation Authors


Trichoptera

Ephemeroptera,

Plecoptera and

Trichoptera

DNA barcoding Gill et al. (2014)

Dinocras cephalotes Plecoptera Genetic diversity Elbrecht et al. (2014)


123

diversity is significant in identification of unique popula-

tions for conservation purpose. In this context, recent

analysis by Taubmann et al. (2011) and Theissinger et al.

(2013) of the genetic population structure of the endan-

gered montane mayfly, Ameletus inopinatus in its European

range is significant. They found decrease of genetic

diversity along an east–west gradient in Central Europe and

along a north–south gradient in Fennoscandia respectively.

Molecular phylogeny and molecular phylogeography

towards conservation: examples from aquatic insects

Molecular phylogeographic studies have major implica-

tions for conservation, e.g. by identifying genetically

diverse populations or communities and/or identifying

potential future refugia of this diversity (Crandall et al.

2000; Hickerson et al. 2010). Furthermore, assessing

genetic diversity in fragmented landscapes among popu-

lations or between sister taxa may contribute to identifying

cryptic biodiversity that is often neglected in conservation

strategies (Moritz and Faith 1998). Maximum-likelihood

methods, which use more of the information contained in

molecular phylogenies in a model-based framework, are

bound to be very powerful as pointed out by Rolland et al.

(2011). Emerging trends in molecular systematics and

molecular phylogeny of mayflies were briefly reviewed by

Sivaramakrishnan et al. (2011). Currently, high-throughput

sequencing, metabarcoding and next-generation sequenc-

ing techniques are contributing to the clearer understanding

of species richness and phylogenetic diversity of various

groups of aquatic insects. Liu et al. (2013) have shown that

the HiSeg 2000 and the SOAP Barcode pipeline a variant

of the genome assembly program SOAP de novo, together

can achieve more accurate biodiversity assessment at a

much reduced sequencing cost in metabarcoding analysis.

Studies of Australian upland stream insects, Cheu-

matopsyche sp., Tasimia palpata and Bungona narilla have

used patterns of variation in the mitochondrial CO1 gene to

examine phylogeographic structure. There was evidence of

past fragmentation followed by subsequent range expan-

sion detected in the nested clade analyses of all three

species. These results indicate alternating contractions and

expansions of available upland habitat during the Pleisto-

cene. During restriction of available habitat, populations of

each of the species are proposed to have become isolated

from one another in upland refugia. Subsequent expansions

of habitat might have caused not only expansions in pop-

ulation size, but also expansions of range and opportunities

for more gene flow, thus causing mixing of previously

isolated lineages (Hughes et al. 2008).

The above examples reveal that much progress have

been made in understanding the evolutionary and

demographic processes that shaped continental biotas

during the Pleistocene. In contrast, the effects of Pleisto-

cene climate change on island biogeography have received

much less attention. Jordan et al. (2005) analysed DNA

sequence data from two closely related endemic Hawaiian

damselfly species, Megalagrion xanthomelas and M. pa-

cificum in order to generate novel insights into the effects

of Pleistocene glaciations and climate change on island

organisms. They found a class of populations (Hawaii

island) that were able to maintain their genetic diversity

throughout glacially induced range shifts, and a class of

populations (Maui Nui) that suffered a loss of genetic

diversity from a series of population reductions, also

associated with global glaciation patterns.

Another significant recent investigation of considerable

conservation value is the assessment of endemism and

diversification in heptageniid mayflies of Malagasy

revealed by coalescent and phylogenetic analysis of

museum specimens and limited field collections followed

by phylogenetic analysis by Vuataz et al. (2013). The

observed monophyly and high microendemism highlight

their conservation importance and suggest the DNA-based

approach can rapidly provide information on the diversity,

endemism, and origin of freshwater biodiversity (Vuataz

et al. 2013).

Identifying potential loss of intra specific genetic

diversity due to global climate change: implications

in conservation

Genetic diversity at the molecular level is crucial for the

maintenance of the evolutionary potential of species and

accurate projections of the future distribution of intraspe-

cific genetic variability which are absolutely indispensable

in the context of increasing evidence for a rapid and pro-

found global climate change (GCC). Recently, Pfenniger

et al. (2012) have presented a methodolical framework

which consists of multiple steps that combine (1) hierar-

chical genetic clustering methods to define comparable

units of inference; (2) species accumulation curves to infer

sampling completeness and (3) species distribution mod-

eling to project the genetic diversity loss under GCC.

Future dimensions

Considerable progress is achieved regarding assessment of

climatically relevant genetic loci as a result of easy and

cost effective access to refined techniques like microarrays,

quantitative PCR (qPCR) and next generation sequencing.

Microsatellites are currently well established as population

genetic markers. However, in conservation genetics,


123

microsatellites are of limited use for identifying ESUs,

management units (MUs), and action units (AUs). In

contrast to PCR-based microsatellite analysis, oligonu-

cleotide probing avoids errors resulting from PCR ampli-

fication. Oligonucleotide fingerprinting generates

individual-specific DNA banding patterns and thus pro-

vides a highly precise tool for monitoring demography of

natural populations. Hence, DNA fingerprinting is power-

ful for distinguishing ESUs, MUs, AUs, and family nets

(FNs). The use of oligonucleotide fingerprinting and fecal

DNA is opening new areas for conservation genetics (Wan

et al. 2004).

To conclude, there is ample evidence to substantiate

how molecular tools help in unraveling the hidden intra-

specific genetic diversity and through this strategy to

address specific conservation issues. As mentioned earlier,

studies that use several molecular techniques to address

related conservation problems provide a more definite

assessment of an issue than those that use only a single

technique. The greatest understanding of molecular infor-

mation for profitable use in conservation of genetic diver-

sity and evolutionary lineages will emerge, however, when

it is used in conjunction with ecological, demographic,

behavioral and physiological data collected in the field

(Haig 1998). Such integrated approach will have serious

implications for the theory and practice of conservation

(Hewitt 2004).

Acknowledgments K. G. Sivaramakrishnan is grateful to Univer-

sity Grants Commission, India for the award of Emeritus Fellowship

[No.F.6-39/2011 (SA-II)] to him to execute his work in Madras

Christian College (Autonomous) with the patronage and support of

Dr. Alexander Jesudasan, Principal and Dr. Arul Samraj, Head,

Department of Zoology. C. Selvakumar thanks UGC, New Delhi,

India for award of Dr. D. S. Kothari Post Doctoral Fellowship

[No.F.4-2/2006 (BSR)/13-670/2012(BSR)]. Authors are grateful to

the anonymous reviewer for critical inputs which have substantially

improved the quality of the manuscript.

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