Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 September 2021 | 13(11): 19566–19579
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
https://doi.org/10.11609/jott.6888.13.11.19566-19579
#6888 | Received 13 November 2020 | Final
received 24 August 2021 | Finally accepted 01 September 2021
Disentangling earthworm taxonomic
stumbling blocks using molecular markers
Azhar Rashid Lone 1, Samrendra Singh Thakur 2, Nalini Tiwari 3,
Olusola B. Sokefun 4 & Shweta Yadav
5
1,3,5 Department of Zoology, Dr. Harisingh Gour
Vishwavidyalaya (A Central University) Sagar, Madhya
Pradesh 470003, India.
2 Department of Biotechnology Dr. Harisingh Gour
Vishwavidyalaya (A Central University) Sagar, Madhya
Pradesh 470003, India.
4 Department of Zoology and
Environmental Biology, Faculty of Science, Lagos State University, Ojo, 102101, Lagos, Nigeria.
1 rashidazhar444@gmail.com, 2 samrendra.thakur01@gmail.com,
3 n.tiwari2987@gmail.com, 4 osokefun@gmail.com,5 kmshweta@gmail.com
(corresponding author)
Editor: Mandar Paingankar,
Government Science College Gadchiroli, Gadchiroli, India. Date of
publication: 26 September 2021 (online & print)
Citation: Lone, A.R., S.S. Thakur, N.
Tiwari, O.B. Sokefun & S. Yadav (2021). Disentangling earthworm taxonomic
stumbling blocks using molecular markers. Journal of Threatened Taxa 13(11): 19566–19579. https://doi.org/10.11609/jott.6888.13.11.19566-19579
Copyright: © Lone et al. 2021. Creative Commons Attribution
4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: None.
Competing interests: The authors
declare no competing interests.
Author details: Azhar Rashid Lone is working
on the phylogenetic systematics and population genetics of Indian earthworms in
natural forests based on COI gene marker. Samrendra Singh
Thakur is working on the metagenomics, soil organic carbon management
(SOC) through soil microbiome and bioremediation of contaminated soils in
Madhya Pradesh, India. Nalini Tiwari is working on the
phylogeny and phylogeography of Indian earthworms
based on COI gene marker.
Olusola B. Sokefun is faculty at Lagos state University, Lagos, Nigeria and expert in
phylogenetic analysis. Prof. Shweta Yadav
is a faculty at Dr. Harisingh
Gour Vishwavidyalaya. She is an expert in molecular
animal taxonomy of earthworms using various molecular markers. She focused her
interest on the monitoring and conservation of fauna in natural forests of
India.
Author contributions: ARL wrote the manuscript, SST and
NT helped in data summarising, manuscript shaping,
OSB helped in mining of phylogenetic data and SY enriched, curated and approved
the final the submission of the final version of the manuscript.
Acknowledgements: Author SST is thankful to
Environmental Planning and Coordination Organization (EPCO), Ministry of
Environment, Govt. of Madhya Pradesh, India for fellowship and SY to Department
of Biotechnology, Ministry of Science and Technology, Govt. of India, New Delhi
for the financial support to carry out the study.
Abstract: Taxonomic classification of
earthworms based on anatomical features has created several challenges for
systematics and population genetics. This study examines the application of
molecular markers, in particular mitochondrial cytochrome oxidase (COI), to
facilitate discrimination of closely related earthworm species. Molecular
markers have also provided insights into population genetics by aiding
assessment of genetic diversity, lineage sorting, and genealogical
distributions of populations for several species. Phylogeography—a
study that evaluates the geographical distribution of these genealogical
lineages and the role of historical processes in shaping their distribution—has
also provided insights into ecology and biodiversity. Such studies are also
essential to understand the distribution patterns of invasive earthworm species
that have been introduced in non-native ecosystems globally. The negative
consequences of these invasions on native species include competition for food
resources and altered ecosystems. We anticipate that molecular markers such as
COI and DNA barcoding offer potential solutions to disentangling taxonomic
impediments in earthworms and advancing their systematics and population
genetics.
Keywords: Annelida, COI, cryptic species,
genetic diversity, Invasive species, Oligochaeta, phylogeography, phylogeny.
Introduction
The terrestrial Oligochaeta include annulated worms known as earthworms or
megadriles, a group of invertebrate animals dispersed all over the world and
having a paramount role in the development of burgeoning soil and its fertility
(Lavelle et al. 1999; Edwards 2004). At present, the earthworms are
investigated all over the world by approximately 300 specialists, most of them
aiming at their ecology and role in terrestrial ecosystems. A few tens of
earthworm scientists are considered to have expertise in Oligochaeta
worm taxonomy and phylogeny. Terrestrial Oligochaeta
has a relatively short and somewhat simple history. Started with the work of
Savigny (1826), the study of earthworms gradually involved more specialists and
consequently became more complicated as new characters and taxa were described.
The contemporary terrestrial oligochaete taxonomy is considered as being rooted
in the classical works of Rosa (1888–1944) and Michaelsen
(1830–1930). Later Pop, Omodeo, Perrel,
Zicsi, and Bouche contributed substantially to the
knowledge of earthworm (especially Lumbricidae)
taxonomy and phylogeny. The studies of earthworms got rapid worldwide
development in the second half of the 20th century with the
development of soil science and soil zoology. Scientists all over the world
were invigorated to study earthworms by the general acceptance of the idea of
the soil, as indispensable for agriculture and must be carefully managed to
avoid its irreversible deterioration. At that time, soil-inhabiting animals
began to be looked as ‘main soil builders’ not only by a few zoologists, but by
a large circle of specialists interested in improvement and conservancy of soil
productivity. Only in a few decades, the main interest of specialists targeted
more and more at the ecological aspects of soil inhabiting animals. More
applicable fields were separated from the theoretical aspects by the processing
of organic materials by earthworms. It also proved to merit protection from the
negative effects of pesticides and even some fertilizers. A large section of
scientists turned their interest to the study of earthworms. But at the same
time, a classical field of earthworm taxonomy and phylogeny didn’t magnify
equally. The novelty and ecological approaches of the animal overlapped their
basic studies. The majority of active scientists turned their interest to the
ecology and application part of earthworms and the earthworm taxonomy was
somewhat neglected or even considered to be outdated. Nevertheless, due to
large-scale faunistic investigations, promoted by the biodiversity and
ecosystem structure investigations, a lot of unknown taxa were found and
described. The scarcity of skilled taxonomists led to the inflation of
improperly described earthworm taxa and the appearance of parallel
classification. Ecologists were firstly affected, but even specialists hardly
succeeded to extricate the entangled stumbling block of earthworm taxonomy. It
became obvious to develop a technology to resolve taxonomic impediments with
the use of molecular tools while the traditional taxonomy and modern molecular
taxonomy have contributed equally to the advancement of earthworm taxonomy.
Traditionally, earthworms
are characterized based on classical approaches like morphological
investigations of the external body and anatomy-based dissections which take
the advantage of limited taxonomic parameters like the structure of prostate,
seminal vesicles, spermatheca, and calciferous glands (Lalthanzara
et al. 2018). Moreover, due to
simplicity of their structural organization, several diagnostic characteristics
in earthworms are inconsistent and overlaps beyond taxon (Perez-Losada et al. 2009), their characterization requires
experts which unfortunately are splurging.
The shortage of discriminatory characters in earthworms was first
divulged by Michaelsen (1900) and consequently
defined these animals as ‘sine systemate chaos’. Thus
in all the domains of earthworm research, the existence of these taxonomic
impediments is responsible for major prejudices. The use of a molecular
approach may be a potential resolution to tackle the stumbling block of
earthworm taxonomy. The use of a standard mitochondrial genetic marker often
termed DNA barcoding has been, nowadays, considered as a reliable approach used
in biodiversity studies as well as in species identification (Hebert
et al. 2003, 2004). Chang et al. (2008) and Rougerie
et al. (2009) have given voice to DNA barcoding as a potential solution to
disentangle taxonomic impediments.
The study reviewed the
prospective of molecular approaches including short sequences of the
mitochondrial genome, in particular, the COI and its
preponderance in resolving the stumbling block of earthworm taxonomy. The
present study accentuates the contribution of this gene marker in deciphering
taxonomic impediments primarily identification of species, phylogeny
re-constructions, intraspecific variations; genetic structure, cryptic species,
lineage sorting, and finally its role in the assessment of invasive species
with phylogeographic tagging (Figure 1).
DNA Barcoding and Clitellate
species identification
Before DNA barcoding earthworm
taxonomy relied on the specific morpho-anatomical features, however, most of
these features often overlap among taxa and it became more inadequate when
recently divergent species or species complexes were entertained (Chang &
James 2011). Although, the allozymes, RAPD, RFLP, and SSR techniques in the
mid-19th century reflected the notion that certain earthworm species
could be segregated. Nonetheless, due to their certain limitations including
dominance and less reproducibility, the focus was given to the use of various
gene markers to gain a better understanding of earthworm taxonomy (Kumari & Thakur 2014). DNA barcode occupies 658 bp of the mitochondrial genome for the recognition of
animal species (Hebert et al. 2003). This method has diverse advantages;
firstly, it is a rapid and cheaper technique in the case of massive samples for
accurate identification. Secondly, it is reproducible and testable since it
always keeps the record between any barcode and its voucher specimen. Above
all, it could be applicable for tissues and applied to any life stages whether
cocoons or a juvenile of any animal species as well as it is accessible
everywhere around the globe (Rougerie et al. 2009).
DNA barcoding has the potential for earthworm research in taxonomy and ecology
(Decaëns et al. 2013). Moreover, in eco-toxicological
studies, it is very essential to identify accurate model organisms for
inferring toxicity of several compounds, as it is evident that many closely
related species can react to the same toxicant differently. Otomo
et al. (2009) highlighted the importance of DNA barcoding for the
identification of earthworm species used in ecotoxicological tests and
concluded that reliable identification is very crucial since it prevents
various discrepancies when comparative studies are done involving different
test species. Similarly, to evaluate the practicability and consistency of DNA
barcoding, an international ring test was organized by Römbke
et al. (2016) who assessed the genetic differentiation of two ecotoxicological
earthworms, viz., Eisenia fetida and Eisenia
andrei. These investigations have not only
assessed the potential of DNA barcoding in taxon identification but specify
that it could be the only way to measure an accurate level of biodiversity (Proudlove & Wood 2003). The study of Richard et al.
(2010) shows the potential of DNA barcoding can be applied to identify juvenile
earthworm species in soils when reference DNA barcode library is available and
thus highlighted that the bias in juvenile collection and identification could
be highly reduced in earthworm biodiversity assessments. Moreover, many
earthworm taxonomists emphasized that integrating morpho-anatomical features
with barcoding data provide more contrasting conclusions. These integrative
approaches were utilized to discriminate among species and taxa that are new to
science (Shekhovtsov et al. 2014; Jeratthitikul
et al. 2017; Lone et al. 2020). Furthermore, compared to
morpho-anatomical features that require exhaustive work, species discrimination
using DNA barcoding is relatively rapid and identification measure is
progressed (Gregory 2005). These in turn have addressed certain issues,
including rehabilitations, synonymies, and description of new taxa. Thus it
sustains the decisions of nomenclature experts and thus primarily contributes
to biodiversity assessments from local to global scales. Therefore, adopting
DNA barcoding has enhanced the accuracy of earthworm studies and in particular,
greatly benefited the community of soil biologists in the description of many
novel species over the past few years (Blakemore 2013; Zhao et al. 2015; Aspe et al. 2016; Csuzdi et al.
2017; Seesamut et al. 2018; Lone et al. 2020); see
Table 1 for more details. Furthermore, DNA barcoding has also shown its
congruent results with other nuclear and mitochondrial genes (Pop et al. 2007;
King et al. 2008) and many such papers are published in peer-reviewed journals.
Furthermore, the nuclear and mitochondrial genes greatly differ in their
divergence rates at different taxonomical levels. In many studies, it has been
inferred that the mitochondrial gene particularly COI has the highest sequence
divergences than other mitochondrial (12S, 16S) and nuclear genes (18S and 28S)
(Chang & James 2011). This indicates that at the species levels or
intraspecific variations, species could be better studied when the
fast-evolving genes like COI are considered. However, at higher taxonomical
levels (within a genus or interfamilial) COI has a relatively weak signal than
other slow evolving genes (18S, 28S) (Chang & James 2011) and should be
used at the species level or within genus if the genus is not too diverse.
Thus, COI has been one of the most influential gene markers which have strongly
revolutionized earthworm taxonomy by avoiding taxonomic confusions and
providing additional evidence for discrimination of taxa over the past few
years.
Role in Phylogeny reconstructions
Dobzhansky (1973) stated that in
biology, nothing makes sense without the consideration of evolution. Since
species undergo evolutionary changes, the relationship of these changes at all
levels provides perception in the phylogenies of diverse species. The
collaboration of morphological and molecular methods has shaped significant
progress in understanding the phylogeny of most major invertebrate groups
(O’Grady & DeSalle 2018). However, this is
partially true for the earthworms which have not been resolved, although many
attempts were made. About 100 years ago and throughout the greater part of the
20th century oligochaetes, sensu
stricto were classified into two main
groups: Megadrila and Microdrili.
The classification was based largely on two parameters; size and habitat
preferences. The larger group that is confined to soils was termed Megadrili and the smaller group that is mostly restricted
to water was called Microdrili (Benham 1890). Later Beddard (1895) compiled the basic structure laid out by
Benham, however, redefined Microdrili by adding the
family Naidomorpha’ (presently called Naididae) - a group that Benham considered as a subclass
distinct from the rest of the oligochaetes. Following cladistic analysis and
reclassification of Oligochaeta, Jamieson (1988)
anticipated a new name for the Megadrili group, Metagynophora, based on the inferred loss of ovaries
located anteriorly. He also proposed Crassicilitellata
a less inclusive taxon for about 3,000 earthworms, containing multi-layered
clitellum (composed of several epidermal cell layers). Whilst, other
oligochaetes app. 120 Metagynophora species that
mostly belonged to the family Alluroididae and Moniligastridae, outside Crassiclitellata,
contained single-layered clitellum. The molecular phylogenetic analysis
although started in the 1990s however, it was not until Siddall et al. (2001)
for the first time focused on the phylogenetic study of leeches and their
relatives that also included earthworms. Later, Jamieson et al. (2002)
published their work on the phylogenetic study of earthworms and revealed
monophyly of the Megascolecidae family based on 12S,
18S, and 16S data, besides it supported the clade Crassiclitellata
(Jamieson 1988). Subsequently, many papers were published on the phylogeny of
earthworms (Table 2). Moreover, to construct a phylogeny in earthworms, the
selection of accurate markers would be essential. COI is
preferred due to its simplicity of primer design and range of its phylogenetic
signal (Hebert et al. 2003), rapid evolution to discriminate at the species
level (Wishart & Hughes 2003), and to provide informative features (Siddall
et al. 2001; Pop et al. 2003; Heethoff et al. 2004;
Chang & Chen 2005; Pérez-Losada et al. 2005;
Chang et al. 2007, 2008; Huang et al. 2007; King et al. 2008). Although many
other genes are taken into consideration for the construction of phylogeny in
earthworms (see Table 2), COI is generally engaged for its rapid divergence and
fast-evolving features that aid in a better understanding of evolution and
phylogeny reconstructions. Irrespective of being a vital role and promising
idea that DNA barcoding has given to the molecular phylogenetics, the ongoing
debates on earthworm systematics still face many key challenges that need to be
addressed (Chang & James 2011). Perhaps, these overwhelming challenges are
not only confined to earthworms but also the whole Annelida. As McHugh (2001)
stated that the poor resolution at higher levels in Clitellata
is due to radiation or rapid divergence of annelid phylogeny and Martin et al.
(2000) stated the same reasons for Clitellata
phylogeny which was also supported by the investigation of Maekawa et al.
(2001) and Su et al. (2001). This demands further research and large datasets
to answer the key questions in Clitellata phylogeny.
Although the molecular phylogenetic investigations were studied in the family Eudrilidae, Ocnerodrilidae, Lumbricidae, Megascolecidae, and Glossocolecidae, however, except for the support of the
monophyly in Megascolecidae, the support for all the
families is weak due to insufficient sampling and taxon bias. Moreover, in the
family Lumbricidae, the focus is given to within
genus (Aporrectodea/Allolobophora, Dendrobaena, and Octodriloides/Octodrillus/Octolasion)
which led to restating the polyphyletic nature of Allolobophora and
synonymizing Octodrilus with Octodriloides, nonetheless, there was no significant
progress in phylogenetic revision (Pop & Wink 2004; Pop et al. 2003, 2007,
2008; Cech et al. 2005). Thus we can anticipate that the phylogeny of the
oligochaetes Clitellata still encompasses various
challenges in the present scenario, and requires further development for
in-depth phylogenetic information. Moreover, DNA barcoding has no doubt
interpreted many findings either alone or with the combination of other genes
however, more data is required to tackle many challenges in phylogenetic
studies in Clitellata and lastly the more densely the
taxa are sampled, the more defined the phylogenetic estimations will be measured
(Erséus 2005).
Unveiling cryptic species/species
complex/intraspecific divergence in Clitellata
In the biological process,
cryptic speciation results in a species group, containing individuals that are
morphologically identical to each other however belong to different species
(Pérez-Losada et al. 2005). With morpho-anatomical
features, most of the cryptic species/species complexes remain unnoticed and it
was not until with the availability of DNA sequences there was an increase in
the number of cryptic species (Torres-Leguizamon et
al. 2014; Marchán et al. 2017). In earthworm
taxonomy, the identification of taxa at higher taxonomical levels particularly
at genus or interfamilial levels can be studied effectively as there are many
taxonomical characteristics that could be applied to assign taxa at family and
genus levels (Pérez-Losada et al. 2005). However,
when closely related species and species complexes are considered, few
morpho-anatomical features are available and it makes taxonomy more complicated
when these morpho-anatomical features overlap among them (Lalthanzara
et al. 2018). Thus at the species level or when dealing with cryptic species,
the taxonomic methods are complicated, exhaustive, labor-consuming,
and demands expertise in the field (Lalthanzara et
al. 2018; Thakur et al. 2020). Furthermore, due to simple body structures in
earthworms, their identification is limited to mature specimens as the key
taxonomical features can only be applied to them, leaving juveniles or closely
related species unidentified. With DNA barcoding several cryptic species/
species complexes are identified in earthworms, most of which are widespread in
several families; Lumbricidae (Heethoff
et al. 2004; King et al. 2008; Fernández et al. 2011; Shekhovtsov
et al. 2013, 2016a), Mediterranean Hormogastridae
(Novo et al. 2010, 2011), Megascolecidae (Chang et
al. 2008; Buckley et al. 2011), Glossoscolecidae (de Faria et al. 2013) respectively (see Table 3 for more
published papers). Moreover, the development of DNA barcoding cryptic species
in earthworms has gained pace as more and more data is being added which not
only tells us the extensive occurrence of cryptic diversity in earthworms but
the action of various ecological processes that has led to these divergences
within them. Furthermore, many investigations revealed that several earthworm
taxa may contain two to five cryptic lineages with app. 10–20 % of nucleotide
substitutions among them (Nova et al. 2009; Buckley et al. 2011; Porco et al. 2013; Fernández et al. 2016). In soil-dwelling
invertebrates particularly earthworms the occurrence of these cryptic lineages
is common due to allopatric isolation which restricts gene flow between regions
of suitable habitat (Hogg et al. 2006) as well as minimizes the change in
morphological characters taking place during speciation (Bickford et al. 2007).
In addition to this, the
different individuals of a given species are not genetically identical. Their
DNA sequences differ to some extent, and these differences form the genetic
diversity, known as the intraspecific diversity of a species (Stange et al. 2020). These genomic variations are the basic
foundation of biodiversity. It refers to a process by which the characteristics
of living organisms change over many generations and addresses how different
species are related through the complicated family trees. Understanding
diversity at the genomic level including an arrangement in taxonomic standards
is, therefore, the most important parameter of biodiversity. The importance of
genetic variation in biodiversity evaluation has been well recognized (Des
Roches et al. 2018). Nonetheless, such studies cannot be accomplished entirely
based on simple morphological examinations of different taxa and therefore
demand molecular investigations to provide more tangible understandings of
earthworm diversity indices. Moreover, molecular studies, for example,
systematic studies involve molecular data to reveal variation among the
population as well as among species. However, molecular systematics rely
largely on empirical results: therefore, increasing knowledge about rates of
nucleotide change is needed to improve assumptions generally used for
phylogenetic inferences and deciphering the evolutionary process within or
between species. While phylogenetic relationships can be deciphered through
analysis of DNA sequences among species, comparisons of DNA barcodes within
species furnish information about the population structure of species and their
evolutionary history.
In earthworms despite their
fundamental importance in soil ecosystems, their population structure as a
function of intraspecific diversity or genetic diversity is poorly understood
and the amount of these studies are scanty, due to either less attention that
was given to earthworms or other vertebrates were studied utmost. Presently
limited investigations such as the role of glacial periods and contemporary
processes like habitat fragmentation on the genetic diversity (see Table 3) of
earthworms are studied based on the partial sequencing of COI gene and other
markers (COII, 12S, 16S, 18S, 28S, H3, H4, tRNAs) and
this has opened up new challenges in the field of population genetics.
Earthworms have a complicated pattern of gene flow with a weak relationship
between genetic and geographic distances. Kautenburger
(2006) studied the genetic structure of Lumbricus
terrestris L populations at different
locations in Germany and revealed an absence of isolation by distance pattern.
Similar observations were inferred by Cameron et al. (2008) while
investigating Dendrobaena octaedra populations in Alberta, Canada. They
pointed out that the anthropogenic activities mainly ‘bait abandonment’ and
limited active dispersal abilities lead to the significant population
differentiation of D. octaedra. These
results are related to the ideas of Sakai et al. (2001) who underlined that
earthworms have limited active dispersal and it is often animal-mediated
transport or limited active dispersal abilities causing genetic differentiation
patterns. The genetic variations in the infields and the outfields of Lumbricus rubellus,
caused by the selection of effective land-use practices (example infield
eutrophication) was studied by Enckell et al. (1986)
while Terhivuo & Saura
(1993) stated that the high clonal diversity of Aporrectoda
rosea is attributed to dispersal activities
through agricultural practices in southern Finland. Terhivuo
& Saura (1997) emphasize that human activities
are the main cause of passive dispersal in Octolasion
cyaneum in northern Europe. Contrary to
these results the reports of Novo et al. (2009) reflected
that Hormogaster elisae contained
cryptic species and the genetic differentiation was primarily based on the
isolation by distance mechanism. The work of Torres-Leguizamon
et al. (2014) on earthworm populations of Aporrectodea
icterica reflected low genetic polymorphism
and that the human-mediated favors dispersal among
geographically distinct populations. Therefore these studies indicate that the
population genetic structure of earthworms is strongly influenced by human
activities. Giska et al. (2015) while studying the
lineages of Lumbricus rubellus of the UK revealed that the mitochondrial
lineages are deeply divergent, however not reproductive isolated and therefore
may constitute a single polymorphic species rather than a complex of cryptic
species. More recently, Ganin & Atopkin (2018) studied the molecular differentiation of two
ecological and three color morphs of Drawida ghilarovi.
They concluded strong genetic differentiation in two ecological forms (anecic and epigeic) with the
presence of several genetic lineages in anecic forms.
The genetic diversity of Amynthas triastriatus populations revealed two genetic
lineages that were split at 2.58 Ma at the time of Quaternary glaciation in
southern China as the authors (Dong et al. 2020) suggested that parthenogenesis
could be an internal factor that influenced the genetic differentiation and
dispersal of A. triastriatus. Taking
together these studies, it can be anticipated that the Clitellata
and in particular earthworms are heterogeneous groups and are prone to genetic
differentiation. The genetic heterogeneity is due to cryptic speciation (King
et al. 2008) or the amphigonic and polyploidy strains
within populations (Casellato 1987). Yet, whatever
the possible reasons that gave rise to genetic heterogeneity, the evolutionary
and ecological consequences of its existence are ranging extensively.
Furthermore, more data is required in terms of COI barcodes along with the
sequencing of other mitochondrial (COII, 12S, 16S) and nuclear genes (18S, 28S)
to understand how earthworms move in soils, how ecological and anthropogenic
activates affect the gene flow and selection in earthworms, and how
environmental stressors are manipulating the genetic differentiation in various
populations of earthworm species. These studies could be essential to
understand environmental changes through these ‘unsung heroes’ of the soil.
Phylogeography and earthworm invasions
Phylogeography is an emerging field that
evaluates the geographical distribution of genealogical lineages. It is based
on the analysis of DNA variations from individuals across a species range to
reconstruct gene genealogies. To infer historical biogeographic events in
species, phylogeography became a potent tool to
understand the role of historical processes in shaping the distribution of
biological species (Avise 2000). It has its role in
invasion biology by improving the knowledge of invasive species. Since, the
speed of invasion has dramatically increased over the past several decades due
to enhanced globalization, as a result of being transported to other continents
via trade either deliberately or unintentionally. This has caused the
transmission of several species to other regions across water bodies where they
usually are absent and now have become recognized beyond their natural ranges
(Hulme 2009). Moreover, once these non-native species invade native terrestrial
ecosystems, they often compete for the resources thus out-competing native species.
This has attracted many ecologists and conservationists to pinpoint their
concerns including alterations in native ecosystems as well threats to the
native species, biodiversity, and economy (Tsutsui et
al. 2000; Pejchar & Mooney 2009; Vilà et al. 2011; Qiu 2015). To
overcome the invasion of these invasive species we not only need to understand
their relationship with native ecosystems in terms of dynamics and
establishments but also the knowledge of the history of their invasion and
ecology. Nonetheless, in some instances, we even do not know the systematics of
these invading species taxa (Yassin et al. 2008; Folino-Rorem
et al. 2009; Bastos et al. 2011) and this makes it more problematic to predict
and manage the invasion issues. Thus, the study of phylogeography
is essential in the sense that it tells us the history of invasive species and
the exploration of their cryptic diversity. Therefore, apart from predicting
its diversity phylogeography helps in the management
of the spreading of invasive species (Schult et al.
2016). Since phylogeography is based on the DNA
sequences of the genome or molecular markers, the variations of patterns in DNA
sequences of these molecular markers leads to the conclusions of how
biogeographic events took place in all geographic scales ranging from
continental to local (Avise et al. 1987; Avise 2000). Moreover, a phylogenetic tree reveals clear
results of how demographic and phylogeographic forces together constitute the
lineage distribution of species. Therefore, to construct a phylogeny and to
depict phylogeography of taxa, the selection of
accurate markers would be essential. Amongst these various molecular markers
the mitochondrial genes (COI, COII, 12S, 16S) especially COI is ideal while
inferring phylogeography and invasion of various
terrestrial species (Chang et al. 2008; Porco et al.
2013; Shekhovtsov et al. 2018a,b). Subsequently, most
of these invasions are taking place in terrestrial ecosystems therefore, it is
vital to understand the ecology, population dynamics of these invading species
before setting management protocols to overcome their ecological effects.
Earthworms being most dominant in terrestrial soils have profound ecological
consequences especially in soils where they actively participate in nutrient
cycling and other soil dynamic functions (Edwards 2004). Since earthworms are
an archaic invertebrate animal group, their phylogeography
is quite restricted due to their little mobilities in soils and incompetency to
cross rivers, seas, and mountains. However, earthworms have been widespread
recently due to two main reasons: via agriculture and commerce carried by
humans across the globe and secondly, the introduction of earthworms in soils
for their effective functions. For example, in the coniferous forests of
Finland (Huhta 1979) Aporrectodea caliginosa was introduced to enhance its
promising results. Similarly, earthworm invasions with their middens and
burrowing activities have no doubt enhance soil heterogeneity and abundance of
other soil invertebrates by creating microhabitats with larger pore sizes and
high microbial biomass that attract micro and mesofauna,
respectively. However, such functions are often transient, small, and
restricted to soil habitats, and rather the invasion has more negative effects.
For instance, the invasion of the Amynthas species
that belong to the Asian Megascolecidae family has
drawn major concerns in the United States and several studies have investigated
their consequences in non-native habitats (Hendrix & Bohlen 2002; Schult et al. 2016). The study of Cameron et al. (2008)
revealed single and multiple invasions of earthworm Dendrobaena
octaedra in the boreal forest of Alberta.
Similarly, Novo et al. (2015b) studied the invasion of Amynthas species
namely A. corticis and A. gracilis in Miguel islands in the Azores. Table 4
provides details of some peer-reviewed papers on phylogeography
and invasion of earthworm species. Thus, in the longer term, the invasion of
non-native earthworms can have strong adverse impacts on native faunal groups.
Other studies either field or laboratory-based investigations also provide
strong evidence of physical disturbance to the soil, food competition,
vegetation loss, alteration of organic horizons, and decline of significant
micro and mesofauna in soils due to invasions (Bohlen
et al. 2004a,b; Frelich et al. 2006). Thus, the
concern of non-native earthworm species should be addressed primarily and more
focus should be given to their population dynamics, cryptic speciation, and phylogeography to understand the network of their invasion
and to overcome their consequences by providing enough unbiased sampling and
DNA based datasets.
Conclusion
Regardless of the fact that
earthworm fauna of India is well reported as compared to other Asian Countries
mainly on the basis of classical taxonomy but to solve a large number of
taxonomic disagreement, an integrated approach of taxonomy may be promising in
this direction. Molecular systematics of Indian earthworms is at nascent
because of limited molecular database. A total of 801 DNA sequences of Indian
earthworm are available on the BOLD database, while limited numbers are
published yet. It is difficult to count them for correct identification unless
they published. In spite of seemingly promising idea of molecular phylogenetic
of earthworms a lack of comparative phylogenetic and phylogeographic inference
have been observed. To overcome the current muddle of taxonomic puzzle of earthworms there is a
need to move on towards integrated taxonomy.
Table 1. List of publications
based on molecular markers in earthworm diagnostics and taxonomy.
|
Marker(s) |
Main focus |
Region(s) |
Reference |
|
COI |
New species (Pontodrilus longissimus) description |
Thailand and Peninsular
Malaysia |
Seesamut et al. 2018 |
|
COI |
Description of new species Eisenia nordenskioldi mongol and Eisenia nordenskioldi onon |
Mongolia |
Blakemore 2013 |
|
COI/ITS |
Aquatic oligochaetes
identification |
Switzerland |
Vivien et al. 2015 |
|
COI/morphology |
New taxa identification |
Kamchatka |
Shekhovtsov et al. 2014 |
|
COI/16S/18S/28S/ H3/H4/tRNAs
|
Description of new
species Eiseniona gerardoi within Lumbricidae |
Extremadura, Spain |
Cosín et al. 2014 |
|
16S/28S/COI/H3/ tRNAs |
Description of new
species Hormogaster joseantonioi |
Teruel Aragon ranges,
Aragon, Spain |
Marchán et al. 2014 |
|
COI |
DNA barcoding of Kanchuria species |
Meghalaya, India |
Lone et al. 2020 |
|
COI |
DNA barcoding of Eutyphoeus species |
Mizoram, India |
Thakur et al. 2020 |
|
COI |
DNA barcoding of earthworms
species |
Madhya Pradesh, India |
Tiwari et al. 2020 |
|
COI |
DNA barcoding |
Thailand |
Jeratthitikul et al. 2017 |
|
COI/16S |
DNA barcoding and phylogeny in
genus Glyphidrilus |
Thailand |
Jirapatrasilp et al. 2016 |
|
COI |
DNA barcoding |
Arunachal Pradesh, India |
Lalthanzara et al. 2020 |
|
COI |
DNA barcoding in Amynthas genus |
Northeastern India |
Vabeiryureilai et al. 2020 |
|
COI |
DNA barcoding |
Uruguay |
Escudero et al. 2019 |
|
COI |
Description of new taxa |
Taiwanese montane |
Chang et al. 2007 |
|
COI |
DNA barcoding |
China |
Huang et al. 2007 |
|
COI |
DNA barcoding |
Taiwan |
Chang et al. 2009 |
|
COI |
DNA barcoding |
Canadian Centre for DNA
Barcoding (CCDB) |
Rougerie et al. 2009 |
|
COI |
DNA barcode for juvenile ID |
Haute-Normandie, France |
Richard et al. 2010 |
Table 2. List of some peer reviewed publications
in earthworm phylogeny and systematics.
|
Marker(s) |
Main focus |
Region(s)/Platform |
Reference |
|
COI |
Phylogeny of Eisenia. nordenskioldi |
Siberian and Korean |
Hong & Csuzdi
2016 |
|
COI/16S/18S/28S/H3/H4/tRNAs |
Hormogastridae phylogeny |
46 sites in the Iberian
Peninsula to Corsica and Sardinia |
Novo et al. 2011 |
|
COI/16S/18S/28S/H3/H4/tRNAs |
Phylogeny reconstruction of Hormogastridae |
Mediterranean |
Novo et al. 2015a |
|
COI/COII/12S/16S |
Earthworm phylogeny genes |
Austria, Canada, USA, Russia,
Croatia, and Ireland |
Klarica et al. 2012 |
|
18S/28S/12S/16S/ND1/COI /COI/I/tRNAs |
Phylogeny of Lumbricidae |
Iran |
Bozorgi et al. 2019 |
|
COI/COII/12S/16S/18S/28S/ ND1/tRNAs |
Evolution of lumbricids |
Europe, USA, Brazil, Africa,
UK, China, Israel, Turkey, and Vietnam |
Domínguez et al. 2015 |
|
28S/12S/16S/ND1/COII/tRNAs |
Lumbricidae phylogeny |
Northwestern Spain |
Domínguez et al. 2017 |
|
COI/16S/ITS2 |
Phylogenetic analysis of
the Dendrobaena byblica |
Balkans, the Greek islands,
Anatolia, Levant and the Carpathian Basin |
Szederjesi et al. 2018 |
|
COI |
Hormogastrid phylogeny |
Iberian Peninsula |
Novo et al. 2009 |
|
COI |
Phylogenetic relationships of
Naidids (Annelida) |
GenBank |
Bely & Wray 2004 |
|
COI/28S |
Monophyly and phylogeny in Eisenia fetida and Eisenia andrei |
Ireland and Spain |
Pérez-Losada
et al. 2005 |
|
12S/16S/28S/COII/ND1/tRNAs |
Phylogenetic relationships
of Aporrectodea caliginosa species complex |
European earthworms |
Pérez-Losada
et al. 2009 |
|
COI/12S/16S/28S/H3/ITS |
Phylogeny of Limnodrilus |
North America, Europe, Japan,
and China |
Liu et al. 2017 |
|
COI/16S/H3/ITS2 |
Cognettia diversity |
Northern Europe |
Martinsson & Erséus 2014 |
|
CO1/CO2/CO3/Cytb/ND5/ND4/16S/ND1 |
Phylogenetic relationships of
15 Pheretima complex |
China |
Zhang et al. 2016 |
|
COI/COII/28S/H3 |
Phylogeny of A. caliginosa
complex |
Europe, UK, USA, Egypt,
Australia |
Fernández et al. 2012 |
Table 3. Depicts the peer
reviewed published literature of cryptic speciation/ species complex/
intraspecific divergence in earthworms.
|
Marker(s) |
Main focus |
Region(s) |
Reference |
|
COI/ morphological
characteristics |
Ecological process and
diversification |
Tropical rainforests of French
Guiana. |
Decaëns et al. 2016 |
|
COI |
Genetic diversity and cryptic
species of E. andrei |
South Africa |
Voua et al. 2013 |
|
COI/16S |
Genetic differentiation and
phylogeny of Drawida ghilarovi |
Russian Far East |
Atopkin & Ganin 2015 |
|
COI/AFLP |
Cryptic lineages in Allolobophora chlorotica, A.
longa, A. rosea, and Lumbricus
rubellus |
British earthworms |
King et al. 2008 |
|
COI/ITS2 |
Genetic variations of Eisenia nordenskioldi
pallida |
Northern Asia |
Shekhovtsov et al. 2016a |
|
COI |
Genetic diversity within A.
caliginosa |
Eastern Europe to the Russian
Far East |
Shekhovtsov et al. 2016c |
|
COI/COII/28S/H3 |
Clonal diversity in A. trapezoides |
Europe, Algeria, Egypt |
Fernández et al. 2011 |
|
COI/16S/28S/tRNAs
|
Genetic differentiation in Hormogastrid earthworms |
Iberian Peninsula |
Nova et al. 2010 |
|
COI/ATP6 |
Lineages of the earthworm Lumbricus rubellus |
Poland |
Giska et al. 2015 |
|
COI/H3 |
Cryptic lineages in L. terrestris, L. herculeus and L.
rubellus |
Northern Europe, USA |
Martinsson & Erséus 2017 |
|
COI/ITS2 |
Genetic variations in Eisenia nordenskioldi
subsp. nordenskioldi (Eisen, 1879)
populations and other lumbricids |
Geographically remote areas of
Siberia |
Shekhovtsov et al. 2013 |
|
COI |
Lineage diversity in L. rubellus |
Britain |
Donnelly et al. 2014 |
|
COI/16S/28S/H3/tRNAs |
Cryptic speciation in H. elisae populations |
Center of the Iberian
Peninsula |
Marchán et al. 2017 |
|
COI/7 microsatellite loci |
Cryptic diversity and geography
of Aporrectodea icterica
populations |
France |
Torres-Leguizamon
et al. 2014 |
|
COI |
Cryptic lineages in Lumbricus terrestris |
Europe, northern America |
James et al. 2010 |
|
COI |
Genetic diversity of E.
n. nordenskioldi |
Southern Urals and eastern
Europe |
Shekhovtsov et al. 2016b |
|
COI/16S/28S/H3/H4/tRNAs |
Genetic variability and
cladogenesis in Aporrectodea rosea and A. trapezoids |
Spain, France, Italy and Algeria |
Fernández et al. 2016 |
|
COI/5.8S/ITS1/ITS2 |
Genetic diversity in Rhinodrilus alatus
and R. motucu |
Southeastern Brazil
savannah |
de Faria
Siqueira et al. 2013 |
Table 4. List of publications of phylogeography and invasion of earthworm using COI and other
molecular markers.
|
Marker(s) |
Main focus |
Region(s) |
Reference |
|
COI |
Genetic structure, and
invasions earthworms and Collembola |
Europe and North America |
Porco et al. 2013 |
|
COI |
Introduction earthworm Dendrobaena octaedra |
Northern Alberta |
Cameron et al. 2008 |
|
COI/16S. |
the invasion history of Amynthas agrestis |
Northern United States |
Nancy et al. 2016 |
|
16S/COI/ND1 |
Systematics and phylogeography of Metaphire
formosae species |
Taiwan |
Chang et al. 2008 |
|
COI/16S |
Historical phylogeography
of Metaphaire sieboldi
|
Japan |
Minamiya et al. 2009 |
|
COI |
Phylogeography of E.n. nordenskioldi populations
|
Russia |
Shekhovtsov et al. 2018a |
|
COI |
Dendrobaena octaedra, Lumbricus rubellus, and Eisenia
nordenskioldi nordenskioldi |
Eurasia |
Shekhovtsov et al. 2018b |
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