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An introduction to the species delimitation and larval-adult association of Chinese Hydropsychidae using independent DNA sequences and adult morphology


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Abstract: The larval forms of most Chinese caddisflies remain undescribed. Hydropsychids deserve very high priority for associating larvae and adults because of their great diversity and biomass in aquatic ecosystems. The conventional approaches to associating larvae with adults include larval rearing and morphological identification of pharate adults. Progress in both methods has been very slow because of the strict microhabitat, diet and water chemistry requirements of the larvae and the rarity of pharate adults. A molecular approach using DNA fragments of both nuclear 28S ribosomal DNA (D2 fragment) and mitochondrial COI is developed for fast and reliable species delimitation and larva-adult association. Species boundaries are delimited based on the congruence between male morphology and phylograms constructed from independent gene sequences. Associations are made with reference to a phylogenetic analysis under two criteria: sequence identity between larvae and adults across both genes or placement of larvae nested within a reference species identified from adult males. This paper provides a detailed background review and introduction to the new methodology. Some of the preliminary results are briefly summarized. Associating larvae and adults of hydropsychids using DNA sequences appears to be promising in terms of reliability and speed. This molecular approach is also expected to be helpful in many problems related to polyphenism across insect groups, such as identification of different life stages, metamorphs, sexual and seasonal morphs, and cryptic species.
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Proceedings of the XIIth International Symposium on Trichoptera, June 18-22, 2006, Bueno-Soria, J., R. Barba-Álvarez, and B. Armitage
(Editors), pp. 355-368. © 2007 The Caddis Press.
An introduction to the species delimitation, larval-adult association of Chinese
Hydropsychidae using independent DNA sequences and adult morphology
1 Department of Entomology, Rutgers University, 93 Lipman Drive, Cook College, New Brunswick, New
Jersey 08901, USA; 2 Department of Ecology, Evolution and Natural Resources, Rutgers University, Cook
College, New Brunswick, New Jersey 08901, USA; 3 Department of Entomology, Soils, and Plant Sciences,
Clemson University, Long Hall, Box 340315, Clemson, South Carolina 29634-0315, USA
Correspondence: Xin Zhou, Fax: 1-732-932-7229; E-mail:
Abstract. The larval forms of most Chinese caddisflies remain undescribed. Hydropsychids deserve very high
priority for associating larvae and adults because of their great diversity and biomass in aquatic ecosystems. The
conventional approaches to associating larvae with adults include larval rearing and morphological identification
of pharate adults. Progress in both methods has been very slow because of the strict microhabitat, diet and water
chemistry requirements of the larvae and the rarity of pharate adults. A molecular approach using DNA fragments
of both nuclear 28S ribosomal DNA (D2 fragment) and mitochondrial COI is developed for fast and reliable
species delimitation and larva-adult association. Species boundaries are delimited based on the congruence
between male morphology and phylograms constructed from independent gene sequences. Associations are
made with reference to a phylogenetic analysis under two criteria: sequence identity between larvae and adults
across both genes or placement of larvae nested within a reference species identified from adult males. This
paper provides a detailed background review and introduction to the new methodology. Some of the preliminary
results are briefly summarized. Associating larvae and adults of hydropsychids using DNA sequences appears to
be promising in terms of reliability and speed. This molecular approach is also expected to be helpful in many
problems related to polyphenism across insect groups, such as identification of different life stages, metamorphs,
sexual and seasonal morphs, and cryptic species.
Key words: Trichoptera, Hydropsychidae, species boundary, larval-adult association, 28S ribosomal DNA,
mitochondrial COI, species barcodes, freshwater biomonitoring.
Hydropsychid larvae and the conventional
associating approaches
Water pollution is rapidly depleting potable water
resources in China. Freshwater biomonitoring involves
identifying the species inhabiting a target ecosystem
in order to provide an ongoing assessment of water
quality that is efficient and cost effective. Caddisfly
(Insecta: Trichoptera) larvae are widely used in
freshwater biomonitoring because of their great
abundance and the wide range of pollution tolerances
among their species. Hydropsychid caddisflies are one
of the most frequently encountered macroinvertebrates
in freshwater habitats. Since hydropsychids have a
dramatically wide range of tolerance values (Lenat
1993), species identification becomes a prerequisite
for biomonitoring. Their application in biomonitoring,
however, has been greatly impeded by the lack of
identified and illustrated larvae, especially in countries
such as China, where there has been limited research
on larval identification.
Most caddisfly species are identified from adult males
because male genitalia are complex, relatively invariant
within species, and diagnostic among species. To be
described and illustrated at the species level, the larvae
have to be associated with the identifiable adults
(usually males). The conventional approaches to larval
association usually involve either larval rearing or
morphological identification of metamorphotypes
(mature pharate adult, larval sclerites, and pupal skin
in the same pupal case) (Wiggins 1996). Both
approaches have significant limitations, which is why
a large portion of the caddis fauna remains
unassociated. Larval rearing is complicated and
inefficient, particularly in some groups such as
hydropsychids, where the larvae generally require very
specific physical and ecological conditions (e.g., water
velocity, temperature, dissolved oxygen, food particles
of specific sizes, etc). Alternatively, metamorphotypes
are relatively rare because they are only available in a
brief period of time (2-3 days each year for a given
species). Because the eclosion timing varies across
species, it is very difficult to collect enough
metamorphotypes for a great number of species in any
given short-term study. Furthermore, female
metamorphotypes are of less use in species identi-
fications because most species identifications require
male genitalia. Even when metamorphotypes are
available, larval identity, inferred from the exuvial
sclerites in the pupal case, is still uncertain if species
with similar color patterns, size, and pupation time
coexist in the same area of the stream (Schefter and
Wiggins 1986).
In addition to their applications in freshwater
biomonitoring, caddis larvae have made contributions
in higher-level trichopteran phylogenetic studies
(Frania and Wiggins 1997; Schefter and Wiggins 1986;
Schuster 1977, 1984; Schuser and Etnier 1978; Scott
1975, 1983; Wiggins 1981, 1996). However, phylo-
genetic analysis was greatly impeded by the un-
associated larvae of several hydropsychid species
(Schefter 2005). Certainly, associating and being able
to identify Chinese hydropsychid larvae will help us
to understand the phylogenetic status of some Oriental
caddisfly genera such as Hydromanicus, Hydatopsyche,
Hydatomanicus, and Trichomacronema.
Current status of Chinese Hydropsychidae larval
Currently, 1,603 hydropsychid species are described
worldwide (Morse 2006) (
database/trichopt/). Yang et al. (2006) recorded 136
hydropsychid species from China, including
Arctopsychinae, which is treated as a separate family by
Genera # species recorded # larvae described2
Subfamily Arctopsychinae
Arctopsyche 81
Parapsyche 80
Subfamily Diplectroninae
Diplectrona 60
Subfamily Hydropsychinae
Cheumatopsyche 19 1
Potamyia 10 0
Hydromanicus 12 0
Hydatopsyche 20
Hydropsyche 10 2
Ceratopsyche 23 3
Mexipsyche 13 0
Herbertorossia 11
Hydatomanicus 10
Subfamily Macronematinae
Macrostemum 10 2
Amphipsyche 41
Aethaloptera 11
Polymorphanisus 41
1 Checklist compiled from that of Yang et al. 2005, where Hydropsyche, Ceratopsyche, and Mexipsyche were treated as subgenera of Hydropsyche;
Arctopsyche and Parapsyche were in Family Arctopsychidae.
2 Including 4 species illustrated in Dudgeon 1999, but without descriptions.
3 Oestropsyche and Trichomacronema are newly discovered in China. One new Trichomacronema species is being described by Sun et al.
Table 1. Known Chinese Hydropsychidae genera1 and associated larvae.
some workers (Gui and Yang 2000; Mey 1997; Nimmo
1987; Schmid 1968). Formal descriptions do not yet
exist for most Chinese caddisfly larvae. Among the 129
known Chinese hydropsychid species, only 13 have
been associated and described (or illustrated), while
the majority of endemic larvae remain unknown (Table
1). Lepneva (1970) described and illustrated 7 common
species shared by the former Soviet Union and China.
Tanida (1987) described the larvae of Hydropsyche
tsudai Tani [synonym of Ceratopsyche orientalis
(Martynov)]. Dudgeon (1999) illustrated a number of
tropical Asian hydropsychid larvae without detailed
descriptions. Among them, Macrostemum fastosum
(Walker), Polymorphanisus astictus Navás,
Cheumatopsyche ventricosa Li and Dudgeon, and
Herbertorossia quadrata Li and Dudgeon are
distributed in Mainland China. The natural history of
only a few species with wide geographic range that
covers China has been studied (Dudgeon 1997;
Kocharina 1997; Tanida 1984, 1985). Among the 18
Chinese hydropsychid genera (Table 1), the larvae of
Hydatopsyche and Hydatomanicus remain
undescribed, both of which are mainly distributed in
the Oriental region. Marlier (1978) described the larva
of Hydromanicus seychellensis Ulmer, which was later
transferred to Hydropsyche by Schefter (2005).
Therefore, formal larval descriptions and illustrations
for the genus Hydromanicus are not available at the
Considering the fact that a significant portion of
Chinese hydropsychid species is still unknown, and
that the natural habitats are disappearing with dramatic
speed in China, a new approach to associating larvae
and adults of Chinese caddisflies, especially
hydropsychids, should be developed as soon as
possible. A rapid molecular method of larval
association, as an addition to the traditional methods,
would significantly accelerate the process of larval
descriptions for a poorly known caddisfly fauna.
Use of DNA sequences in species diagnosis and life-
stage association
DNA sequences have been widely used in species-level
diagnosis in a variety of insects, such as beetles (Miller
et al. 1997, Peng et al. 2002), parasitic wasps (Babcock
and Heraty 2000, Tilmon et al. 2000), and a number of
dipterans with important forensic or medical
significance (Fettene et al. 2002, Huong et al. 2001,
Marrelli et al. 1999, Somboon et al. 2001, Toma et al.
2000, Vincent et al. 2000, Wallman and Donnellan
2001, Wells et al. 2001). A protocol of using partial
mitochondrial COI sequence (i.e., DNA barcodes) in
species identification has been developed (Hebert et
al. 2003a, Hebert et al. 2003b, Hebert and Gregory
2005) and has shown its potentials in differentiating
closely-related insect species as well as improving
biodiversity inventories (Ball et al. 2005, Hajibabaei
et al. 2006, Hebert et al. 2004, Janzen et al. 2005).
Although DNA sequences have had some success in
associating life-stages (a subcategory of species-level
organism diagnosis) in some invertebrates, few such
examples were insects. Four hundred twenty five
nucleotides of partial mitochondrial leucine tRNA and
COII supported the association of some first-instar
nymphs to their adults in a gall aphid species and
provided evidence to a new hypothesis of host-
alternating life cycle in aphids (Aoki et al. 1997).
Mitochondrial COI/COII/leucine-tRNA sequences of
2300 nucleotides were used to identify the larvae of
three blowfly species to improve their applications in
estimating postmortem intervals in forensic
entomology (Sperling et al. 1994). Wells et al. (2001)
developed a COI database (an idea also held by DNA
barcoding workers) in order to identify fleshfly species
that are likely to be found feeding on human corpse
including both adults and larvae. Shan et al. (2004a,
b) associated 5 Chinese caddisfly species (1 hydro-
biosid, 4 lepidostomatid species) using mitochondrial
COI/COII/tRNA sequence. And recently, a similar
work (Miller et al. 2005) associated a larva to the adults
of a dytiscid beetle Philodytes umbrinus (Coleoptera:
Species boundary and delimiting method
A clear statement of the species concept and a defined
delimiting method are critical in interpreting species
boundaries using DNA sequences. Because a particular
DNA sequence is shared among all life-stages and
phylogenetic relationships can be inferred from these
DNA sequences, a history-based phylogenetic species
concept (Baum and Donoghue 1995) is employed in
this study. Under this species concept, conspecifics
(individuals of the same species) are more closely
related to each other than to any members of other
species. In terms of nucleotide variation, members
within species are expected to possess fewer changes
among themselves than among individuals of other
species. However, delimiting species boundaries based
solely on mean genetic divergence can be arbitrary and
therefore problematic.
In the majority of existing works, a single DNA
sequence (or linked sequences with interdependent
histories) collected from morphologically distin-
guishable specimens of a certain life stage, such as
adult males, or late-instar larvae (e.g., some mosquitoes
and mayflies), is used as a reference. Once the
unidentified individual of the alternative life-stage is
sequenced, a comparison of this test sequence to the
reference may provide an association under certain
criterion of species delimitation. Although not always
explicitly expressed, species boundaries are often
Species delimitation of Chinese Hydropsychidae using independent DNA sequences and adult morphology
delimited by the overall genetic similarity of the test
sequence to the reference sequences. An empirical
average divergence value (“threshold”) is given to
define inter- and intraspecific boundaries. For instance,
among several sarcophagid flies and their close
relatives, mitochondrial COI sequences diverge less
than 1% within species, while greater than 3% among
species (Wells et al. 2001). This putative species
boundary may be tested and corroborated subsequently
with statistic support, such as bootstrap values (e.g.,
Miller et al. 2005).
Some studies on DNA divergences have proposed that
there is some typical range of genetic divergence
among and within species. For instance, mitochondrial
COI sequences diverge greater than 2% in 98% of the
animal congeneric pairs (Hebert et al. 2003b), while
intraspecific divergences of mitochondrial DNA are
rarely greater than 2% and most are less than 1% (Avise
2000, see Hebert et al. 2003b). However one should
be cautious when applying these “typical” thresholds
to other taxa because genetic divergence among species
varies across taxa, a set genetic distance that typically
defines species boundaries in one group may not be
applicable to others. This is particularly true when some
taxa evolved rapidly, forming diversified cryptic
species complexes, while others remain less speciose.
In an extreme case, 88.2% of cnidarians show less than
1% interspecific divergence in COI sequence, while
93% of hymenopterans show divergences in a range
of 8-16% (Hebert et al. 2003b). Hence species
boundaries based on genetic distance have to be
defined specifically for various taxa, and rate changes
can occur at any point.
Alternatively, some works have employed a threshold
generated from the dataset that is being used in the
same study. For example, larva and adults of a dytiscid
beetle have an average pair-wise p-distance of 0.09%,
which is then compared to an average interspecific
divergence of 13.49% among related species (Miller
et al. 2005). The average interspecific divergence,
however, can be greatly reduced through a more
extensive taxa sampling. Indeed, two species from the
outgroup in the same study have an average of 1.9%
divergence, both clades of which have high boot strap
values (95% and 98%, respectively). Moreover, if the
focal species was paraphyletic or polyphyletic, the
intraspecific divergence is dependant on the degree of
polyphyly. For instance, the intraspecific sequence
divergence of mitochondrial sequences in a
polyphyletic leaf beetle Neochlamisus cribripennis
changed dramatically from 0.3% to 10.0% where
different sampling strategies were taken (Funk and
Omland 2003). Paraphyly/polyphyly may not typically
be detected if only a single gene is used in the study,
an issue we will discuss in more detail later. More
importantly, since genetic distance criterion has to be
made from existing data, the logic is circular. The
species boundary should be defined or supported by
sources other than the DNA sequences themselves, e.g.,
an independent gene and/or morphology.
Furthermore, the typical genetic divergence threshold
of a single gene may not provide enough resolution to
differentiate closely related taxa, especially the
youngest sister species (Hebert et al. 2003a, Hebert
and Gregory 2005). Potential random lineage sorting
of ancestral polymorphisms and introgressive
hybridization can complicate diagnoses even further.
For instance, several Japanese carabid beetles have
shown frequent sequence identity across species in
mitochondrial ND5 mainly due to repeated
introgressive hybridization. Previous taxonomy
inferred from an ND5 phylogeny was based on an
artifact of hybridization and gene introgression (Sota
et al. 2001). Although the occasional sharing of
mitochondrial sequences across species may have
limited impacts on the large-scale organism diagnosis
in the DNA barcoding initiative (Hebert and Gregory
2005), it can lead to incorrect larval-adult association,
particularly in closely related species.
Using independent genes to detect species-level
Individual genes frequently have multiple copies
within species, and sometimes ancestrally polymorphic
genes can randomly sort into patterns that do not track
phylogenies or species trees. Species-level polyphyly
(sensu Funk and Omland 2003), where conspecifics
are not monophyletic on a particular gene tree, may
cause discrepancy between gene lineage history and
organismal history. A number of studies have stressed
the consequent impacts on above-species-level
molecular phylogenies, i.e., “gene tree/species tree”
phenomena (Avise et al. 1983, Pamilo and Nei 1988,
Doyle 1992, Brower et al. 1996, Maddison 1997,
Nichols 2001). The causes of this problem have been
discussed intensively by many authors (Gomez-Zurita
and Vogler 2003, Maddison 1997, Nichols 2001, Page
and Charleston 1997, Pamilo and Nei 1988, Simmons
et al. 2000). Additionally, the discordance between
gene history and organismal history will not only bias
conclusions in higher-level phylogenies, but also
endanger species diagnosis. Individuals of the same
species may be grouped together with those of a
different species while their conspecifics are nested
outside. Clearly, in polyphyletic species, the typical
genetic threshold cannot delimit species. A literature
survey of species-level phylogenetic studies involving
mitochondrial DNAs (genes used predominantly in
species-level works) has indicated that polyphyly
(including “polyphyly” sensu stricto and “paraphyly”)
are not as rare as we have hoped. An overall 23% of
2319 study species in the survey exhibited polyphyly
or paraphyly, while 15% of insect species are not
monophyletic (Funk and Omland 2003). Consequently,
species-level polyphyly has to be detected during
species delimitation in order to define confident species
boundaries. Among many others, random lineage
sorting of ancestral polymorphism and introgressive
hybridization are the two major causes of species-level
polyphyly (Funk and Omland 2003). These phenomena
can sometimes be detected by using independent genes.
Individuals of different species may possess more
closely related gene lineages due to random sorting
and retention of ancestral polymorphisms. It may be a
common phenomenon in rapid species radiation, where
speciation events occur before the completion of
lineage sorting (Avise 1994, Maddison 1997).
Consequently, the phylogenetic structure constructed
from any single gene may not reflect the species
boundary correctly no matter what analytical method
is used. The random sorting and fixation of ancestral
polymorphism may be detected by using independent
genes because multiple genes with independent
evolutionary histories will not necessarily share an
identical gene-sorting pattern. Thus a species boundary
delimited by one gene can be consistent (or
inconsistent) with the boundary delimited by another
independent gene. As a result, the possibility of having
an incorrect species boundary caused by differential
sorting of ancestral polymorphism is reduced by
sampling multiple genes.
In addition, although not as common as in plants,
hybridizations have been reported in a variety of
animals, including some insects (e.g., Ballard 2000,
Sota et al. 2001). Surprisingly, insect genitalic
structures, the complicated “lock-and-key” complex
that is expected to prevent interspecific hybridi-
zation, may not always be able to completely prevent
gene flow (Sota et al. 2001). Generally, male
caddisflies have extremely complicated external
genitalia that are species-specific. Even so, the
anatomical differences among some closely related
species can be very subtle, such as in some Chinese
Mexipsyche and Cheumatopsyche species. Because
hybridization often leads to parallelism between the
hybrid and the parents that can obscure the underlying
hierarchy (Funk 1985), failure to detect hybridization
may lead to incorrect species boundaries. For instance,
in Fig1b, species A and B in the clade 1 (haplotypes
A1/A2/A3/B9/B10/B11) appear to be mixed together,
which may have been the consequence of hybridization
between parental species A and B. If for some reason
adult males of species A (haplotypes A1-A3) were not
sampled, a larval specimen that belongs to species A
(larvaA) may appear to be most closely related to B9-
B11. Thus this larva will be associated with species B
Species delimitation of Chinese Hydropsychidae using independent DNA sequences and adult morphology
Figure 1. Hypothetical species boundaries and problematic association
Adults from species A and B are identified based on morphology before a phylogenetic analysis. Species boundaries
are then mapped on the gene phylogram constructed from a DNA sequence. a). Species boundaries without
paraphyly/polyphyly: both species A and B are well delimited; b). Species boundaries with paraphyly/polyphyly:
adult individuals from both species mixed together in clade 1; c). Problematic association: if adults of species A
were not sampled in an incomplete sampling, a larval specimen of species A (larvaA) will be associated incorrectly
with species B.
rather than with species A. The best solution to this
problem would be following a well-designed sampling
strategy that includes an established and growing
database. However, if sampling is relatively complete,
incongruence between independent genes may help to
detect potential hybridization (as well as lineage
sorting), suggesting a need for caution with this
particular association.
Although random lineage sorting of ancestral
polymorphism and retrogressive hybridization are very
different evolutionary processes, the patterns of
consequent haplotype distribution on the phylogram
can be very similar in empirical studies. Theoretical
and statistical methods have been proposed to
distinguish these two as well as many other causes of
gene incongruence (Maddison 1997, Sang and Zhong
2000). Nevertheless, determining the specific reason
that has caused the species-level polyphyly is beyond
the scope of this study. After all, our purpose in this
work is not to clarify the species boundaries of Chinese
caddisflies using DNA data, but rather to make
relatively confidant associations of larvae and adults.
Once the conflicts between independent genes and
morphology are identified, special attention should be
given to particular problematic species. It is better to
draw attention to problematic species, but leave them
undescribed than to make the wrong association. A
well-designed sampling program is necessary in order
to provide convincing data to confirm the causes of
polyphyly. Different analyses should be used for
lineage sorting and hybridization (Vriesendorp and
Bakker 2005, Sang and Zhong 2000).
Gene choice
Sequencing multiple independent genes may permit the
detection of species-level polyphyly. Moreover,
because no gene is ideal for all purposes, independent
genes may complement each other in terms of the
information they provide. For instance, nuclear genes
are less prone to be affected by introgression than
mtDNA (Avise 1994), while the latter suffer less
recombination and gene-duplication (but see
Ladoukakis and Zouros 2001, Smith and Smith 2002).
In the case of random sorting of ancestral
polymorphism, mitochondrial DNAs may be a better
reflection of species history (Moore 1995, but see
Hoelzer 1997, Moore 1997) because of their rapid
coalescence times. On the other hand, nuclear genes
have shown more congruence with morphology in the
case of hybridization (Sota and Vogler 2001). Yet all
current life-stage associating works and many species
diagnosis works rely on a single gene or linked genes
with dependent histories (for example: mitochondrial
COI/COII/tRNA), which in turn should be treated as a
single gene for tracing gene histories. In this paper,
we propose to use two independent gene fragments:
one from mitochondrial COI and a second from nuclear
28S ribosomal DNA, to construct a phylogenetic tree
from which the species boundaries and association of
larvae and adults are made.
Mitochondrial COI gene: Mitochondrial genes
(mtDNA) are most frequently involved in species-
level works. The rapid coalescence, high copy
number, lack of introns, and availability of universal
primers in most animals seem to be the major
advantages over other genetic makers in species
diagnosis as well as phylogenetic studies. Because
mtDNA are maternally inherited, they are rarely
affected by recombination and paralogy/orthology
problems, although recombination of mtDNA in some
animals has been discussed (e.g., Ladoukakis and
Zouros 2001, Smith and Smith 2002) and the presence
of mitochondrial pseudogenes in the nucleus
(Antunes and Ramos 2005, Hay et al. 2004, Schmitz
et al. 2005, Simon et al. 1994, Villegas et al. 2002)
may sometimes result in false sequence readings.
Like most protein-coding genes, mitochondrial
protein-coding genes have few insertions and
deletions (usually none among closely related
species), reducing alignment problems. Further-
more, mtDNA haplotypes have smaller effective
population size (Ne). In theory, their coalescence time
is only ¼ that of nuclear genes (Palumbi et al. 2001,
but see Hudson and Turelli 2003). Because mtDNA
genomes are present in multiple copies, they are much
easier to amplify by PCR than single-copy nuclear
protein coding genes. Numerous mtDNA primers have
been described, such as by Simon et al. (1994). Among
mitochondrial protein-coding genes, the cytochrome
oxidase I gene (COI) is perhaps the most commonly
sampled, and has been selected in the DNA
barcoding project for its robust primers and
relatively conservative amino acid composition
(Hebert et al. 2003a). In this study, COI is easily
amplified with primers developed specifically for
caddisflies in Kjer et al. (2001).
D2 expansion fragment of 28S nuclear ribosomal DNA:
We also include an independent sequence, D2
expansion fragment of 28S nuclear ribosomal DNA
(nrDNA). Nuclear ribosomal DNA (the nuclear gene
that codes for ribosomal RNA) belongs to a multi-gene
family, where hundreds to thousands of copies of the
rDNA unit appear tandemly along chromosomes.
Unlike other nuclear genes that are also exposed under
recombination, numerous copies of a nrDNA unit
throughout the entire genome become homogenized
very rapidly under molecular drive (Dover 1984).
Therefore the effects of paralogy, where genes without
strict orthologous relationships are being compared,
have been minimized due to this concerted evolutionary
D2 expansion fragment of 28S rRNA (or the “545
region” of Schnare et al. 1996) is one of the most
highly variable regions in eukaryote ribosomal
RNA. This fragment, especially the main helices,
is highly variable in length as well as nucleotide
composition among insects (Gillespie et al. 2004).
These significant variations limited their uses in deep
level phylogenetics because of the difficulties in
aligning and assigning homology, although manual
alignment based on RNA secondary structures have
provided solutions for some taxa (e.g., Gillespie et al.
2004). However, length variation is not a severe a
problem in closely related species. Although large
insertions or deletions could potentially be
encountered at very close levels, based on our
preliminary results in caddisflies, the length of the
D2 fragment is very conservative within the genera
we have sampled. The changes in the hypervariable
regions, even when they cannot be aligned across
distantly related taxa, provide opportunity to
differentiate closely related species. In fact, the D2
fragment provided sufficient genetic variation to
distinguish two species of Encarsia wasps where
morphology had encountered extreme difficulty
(Babcock and Heraty 2000) and we will show that D2
is able to distinguish closely related species of the most
speciose Hydropsychidae subfamily, Hydopsychinae.
Furthermore, the highly conservative core segments
that flank the D2 fragment serve as ideal anchor points
for primers. In fact, the D2 primer pair — D2up4 and
D2dnB (see Table 2), has been extremely efficient
across almost all hydropsychids as well as most
caddisflies and other insects.
the same genitalic structures, and are part of a
monophyletic group on the phylogram, are considered
to be putative species. If these putative species
boundaries are the same on each of the two independent
gene phylograms, a working species boundary has been
established. Thus, species boundaries are defined
morphologically, and their monophyly is supported
with the molecular data.
The application of morphology to confirm the species
boundary is critical because: 1) The genes may not be
able to reveal the real history of speciation due to the
properties discussed previously, 2) Because species
boundaries are mapped on the DNA phylogram using
morphology, genetic divergence values are not
involved in defining species, 3) The morphology
provides a third independent reference to species
We propose a molecular approach integrated with
morphology to delimit the species boundary:
1) Construct phylogenetic trees based on
independent analyses of both the D2 and COI
gene fragments collected from adult males
2) On the phylograms, delimit tentative species
boundaries based on male genitalic morphologies
3) Compare the two gene trees:
a) If a tentative species boundary is mono-
phyletic on both trees, this particular species
boundary is well delimited (Fig. 2);
b) Alternatively, if polyphyly appears on one or
both of these trees within a tentative species
boundary (Fig. 1b), the species delimitation
cannot be determined at the moment. A more
complete sampling is required to clarify the
specific cause of the polyphyly for consequent
treatments to be taken, but a growing database
can be established, to which additional
samples can easily be added.
Larval-adult association criteria
Once the species boundaries (based on male adults)
are delimited, larval sequences can be placed into
the analysis. The association is made from the
resultant phylograms. The criteria of associating
larvae and adults are established based on the
topological relationship between larvae and adults.
In the following schemes, the reference species
boundary is represented by adult 1 and adult 2, the
representatives of the most distant individuals in the
species clade, for example A1 and A6 in Fig. 2a, or
A2 and A5 in Fig. 2b. Many other adults could nest
within adult 1 and 2. We expect 3 different
1) One or more larvae are identical to at least one
of the identifiable adults across both genes
(sequence identity, Fig. 3a);
Species delimitation of Chinese Hydropsychidae using independent DNA sequences and adult morphology
Primer Sequence (5’ to 3’)
Table 2. Some PCR primers used in this study.
Delimiting species boundary based on phylogenetic
In this study, the species boundary is defined both
morphologically and phylogenetically, with
morphological characters mapped upon phylogenies
constructed from D2 and COI data collected from
identified adult males. Individuals with the same
genitalic characters are categorized before the
molecular phylogenetic analysis. Once the phylogeny
is established, these morpho-species are mapped upon
the phylogeny. In other words, all members that share
2) Larva nested within the reference species (Fig.
3) Larva placed outside of a reference species.
Both 1) and 2) are considered as being successful
association. More individuals are needed if larval
sequences do not nest in reference species (Fig. 3c).
In most case, the desired added taxa would be adult
males from wider geographical range.
Molecular protocols
Specimen collecting: The majority of larval and adult
specimens in this work were collected from 6 provinces
of China: Guangdong, Guangxi, Jiangxi, Sichuan,
Yunnan and Beijing, from 2001 to 2005. Hydropsychid
larvae were collected using kick-nets and D-nets or by
examining the substratum. Adults were collected by
light-trap and sweep-net. Both larval and adult
specimens were cursorily sorted after collecting and
preserved in 95% ethanol. The ethanol was replaced
within a few days after collecting and changed again
in approximately 30 days. Larvae and adults were
sorted into morpho-species as soon as possible
(typically within 6 weeks) and individuals were
preserved separately. Vouchered larval specimens are
temporarily deposited at Rutgers University, New
Jersey, USA, and will be deposited permanently in
Nanjing Agricultural University, Nanjing, China, upon
completion of the project. The majority of adult
specimens are deposited at Nanjing Agriculture
University while parts of them were used for extracting
DNA at Rutgers.
Abdominal segments 3-6 or legs of larvae and adults
are used for DNA extraction. Larval intestines and gut
contents are removed carefully to reduce the potential
for contaminants. The rest of the specimen was
preserved in ethanol for morphological study.
Total genome extraction: Qiagen DNeasy Tissue Kit
was used to extract genome DNA. The total DNA was
then amplified with PCR using Qiagen Taq PCR Core
Kit or Qiagen Taq PCR Master Mix Kit.
PCR: Primers are designed and ordered from Operon.
For the D2 fragment, D2up4/D2dnB primer pair yielded
the best success. For COI, 1709Fs/2191R worked well
for most taxa; 1709Fg, 1751F and 2209R, together with
other primers were also frequently used (Table 2).
Degenerate primers were designed for some taxa.
Figure 2. Delimiting species boundary based on gene and morphology congruence
Species A and B are identified based on morphology before phylogenetic analysis. Species boundaries are in
tern confirmed by gene congruence across independent COI and D2 sequences.
The PCR reaction mix consisted of 12.5μl Qiagen Taq
PCR Master Mix, 1-4μl of each primer (50pmol/μl),
1μl genomic DNA and up to 9.5μl of ddH2O. The
volumes of primers are adjusted according to the
redundancy. The PCR mix was preheated at 94ºC for 3
minutes followed by 40 cycles of 94ºC for 30s, 60ºC
(with D2Dup4/D2dnB) or 53ºC (with COI 1709Fs/
2191R) for 45s, 72ºC for 60s. After 10 minutes of final
extension at 72ºC, the products were maintained at 4ºC.
PCR products were cleaned using the Qiagon QIAquick
PCR Purification Kit. When multiple DNA bands were
amplified, PCR products were electrophoresed on a
1.5% low-melting point agarose gel and then excised.
The target DNA contained in the gel slice was then re-
covered using a Qiagen QIAQuick Gel Extraction Kit.
Sequencing: DNA sequencing was performed on an
ABI 3100 Autosequencer. A 10μl cycle sequencing
reaction mix contained 2μl 5X ABI BigDye Buffer, 1μl
ABI BigDye v3.1, 1μl primer of 3.2pmol/μl
(concentration adjusted accordingly upward when
using degenerate primers), 1-6μl purified PCR products
which contained 5-20ng of DNA and 0-5μl ddH2O
accordingly. An amplification program was performed
for 30 cycles of 96ºC for 15s, 50ºC for 10s, 60ºC for 4
minutes and held at 4ºC. The products were cleaned
by ethanol precipitation. DNA was re-suspended by
15μl Hi-Di Formamide and incubated at room
temperature for at least 15 minutes. The plate was
heated at 95ºC for 2 minutes and quenched immediately
on ice, and then sequenced.
Sequence editing and aligning: Each individual DNA
fragment was sequenced from both directions.
Sequences from both directions were then aligned and
proofread using program ChromasPro v1.2 (Windows)
or ABI Prism Sequence Navigator 1.0.1 (Mac OS). Any
conflict or ambiguous reading was given one of the
appropriate IUB symbols: Y, R, S, W, K, M and N.
Usually the use of these ambiguity codes is not
indicative of a real polymorphism, but rather, problems
with unambiguously reading the peaks on the
chromatograph. Therefore, when we had an ambiguity
in one taxon, and a defined nucleotide in another that
nests within that ambiguity code, we considered these
sequences to be identical. COI sequences were aligned
using ClustalX v1.83 (Thompson et al. 1997) and
MacClade v4.08 (Maddison and Maddison 2005). D2
fragments were manually aligned in Microsoft Word
referring to the secondary structure (following Kjer
1995). Manual aligning was performed in order to serve
our other purpose of higher-level phylogenetics.
However, it was not necessary for closely related
species. Multiple D2 sequences can be first aligned in
ClustalX and then adjusted by eye. The D2 alignment
is available from the authors and on Kjer’s website.
Primer regions were eliminated from the final
sequences, which yields 439 bps in COI and ~430 bps
in D2, the latter of which has shown significant length
variation in the family Hydropsychidae.
Phylogenetic analysis
Phylograms were constructed independently from both
D2 and COI sequences using distance and neighbor-
joining in PAUP*4.0b10 (Swofford 2003). Distance
parameters were obtained as follows: DNA distances
using Kimura 2-parameter model; missing and
ambiguous data were ignored for pair-wise
comparisons; all substitutions were estimated or
counted; distance criterion were set to minimum
evolution. K2P model was used to take into account
the transition and transversion changes.
Pair-wise distances, within- and between-species
divergences of COI nucleotides were calculated in
MEGA v3.1 (Kumar et al. 2004) in order to provide
comparisons to other species diagnosis works.
Bootstrap values were calculated in PAUP*4.0b10
using neighbor-joining searching type for 1000
replicates; groups with frequency greater than 50%
were retained.
A brief summary of the preliminary results
The detailed results and discussion are being published
separately. Larval descriptions and illustrations are
Species delimitation of Chinese Hydropsychidae using independent DNA sequences and adult morphology
Figure 3. Association criteria.
being organized into independent papers. We only
provide here a summary of some of the preliminary
results and a general prospect of this new larval-adult
association approach.
(1) Both nuclear 28S rDNA D2 expansion fragments
and mitochondrial COI fragment can provide
good resolution to differentiate closely related
species in the Chinese hydropsychids.
(2) COI sequence revealed some intraspecific
divergence that were beyond the typical genetic
“threshold”, thus one should be extremely careful
when interpreting species boundaries in
caddisflies based merely on genetic divergence
(3) Our species delimiting method that has
integrated conventional morphology and
independent gene evidence was able to clarify
species boundaries of the vast majority of
Chinese hydropsychids with few exceptions
due to potential species-level polyphyly or
imperfect taxonomy.
(4) Both morphologic and molecular evidence
revealed that a significant portion of Chinese
hydropsychid species were undescribed, calling
an urgent need for a thorough survey on the
Chinese fauna.
(5) Distance-based algorithms, such as neighbor-
joining, can reflect the species boundaries
between closely related species, although it is
not necessarily informative on deep-level
(6) Associating larvae and adults of hydropsychids
using DNA sequences appears to be promising
in terms of reliability and speed. As an addition
to the conventional association approaches, the
molecular method will greatly improve our
knowledge on caddis larvae.
(7) To date, the cost of the entire sequencing
protocol can be controlled around five USD
per specimen in the best equipped molecular
laboratory (
BarCoding.htm). We anticipate that this cost
will continue decreasing as DNA sequences
are more and more involved in species- and
higher-level phylogenetic studies.
(8) At the moment, approximately some 30
Chinese hydropsychid species, including all
Chinese genera except Oestropsyche and
Aethaloptera, have been associated. As we
gain knowledge on larval taxonomy, ecology
and biology studies can be conducted for the
associated species. In particular, tolerance
values can be assigned to specific species.
Consequently, the opportunity to use
hydropsychid larvae in biomonitoring China’s
aquatic habitats is greatly improved.
We appreciate Sun Changhai of Nanjing Agricultural
University, China, for his important works on
morphological identification of the majority of
Hydropsychidae specimens encountered in this study.
Professor Yang Lianfang and Sun Changhai of Nanjing
Agricultural University and Christy Jo Geraci of
Clemson University all have been extremely important
in organizing and conducting our hydropsychid
explorations in China. This work was supported by the
National Science Foundation (NSF DEB-0316504).
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... In both species they differed solely in one nucleotide. The association of larvae and adults are not completely reliable based solely on comparisons of sequences from one specimen each (e.g., Zhou et al. 2007), but other data (distribution of the D. bosnicus Group in Bosnia and Herzegovina, morphological features of larvae, rearing in laboratory) confirm our association of larvae and adults for both species D. ramae and D. medianus. ...
... The association of caddisfly larvae to particular species with the help of molecular markers is becoming more common in different families (e.g., Zhou et al. 2007) also including Drusinae (Graf et al. 2005, Waringer et al. 2007b, 2008b. Thus, the current study represents a further example in emphasising the importance of molecular markers in supporting taxonomic research. ...
Full-text available
In this study we present morphological features of the last instar larvae of Drusus ramae Marinkovic-Gospodnetic, 1971, and Drusus medianus Marinkovic-Gospodnetic, 1976, both of which are endemic species from Bosnia and Herzegovina. Using the mitochondrial cytochrome oxidase I gene (COI), larvae of these species were successfully associated with adult specimens. We present the most important diagnostic features enabling separation from larvae of the other Drususspecies and an identification key for the known larvae of Drusus species in the Balkan Peninsula. Notes on the distribution and ecology of both species are given. Additionally, faunistics and possible conservation implications for studied springs are discussed.
... The analysis shows that COI sequence clusters were recovered for the two morphological species analyzed in Glossosoma. Female, larval, and pupal specimens were associated with adult males for G. kamael according to the criteria outlined by Zhou et al. (2007). The maximum genetic distances of all specimens in the G. kamael cluster were less than 0.48%. ...
The adult female, larva, and pupa of Glossosoma (Lipoglossa) kamael Malicky 2012 from Qinghai Province, China, are described and illustrated. Molecular associations are based on COI sequences. The immature stages of the subgenus Lipoglossa are described for the first time. Biological and habitat information of G. kamael are presented.
... Female, larval, and pupal specimens were associated with adult males for L. deqianensis n. sp. according to the criteria outlined by Zhou [40]. Their COI sequences were uploaded to GenBank and their accession numbers are included in Table 3. ...
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Fifty individuals of Limnephilus from the Qinghai Province, China, were examined, and their COI barcode sequences were extracted and analyzed. Forty individuals of Limnephilus from the Insect Collection of Nanjing Agricultural University (ICNAU), China, were examined, and photos of the male genitalia of four Limnephilus species are here presented. The males, females, larvae, and pupae of a new species, Limnephilus deqianensis n. sp., associated via COI barcode sequences, are described and illustrated. Ecological photos of the male, pupal case, and the habitat of the new species L. deqianensis n. sp. are also provided. Five species groups containing all seventeen Chinese Limnephilus species are revised. Diagnoses, keys, and a distribution map of them are provided. All of the sequences have been uploaded to GenBank. All specimens are deposited in the ICNAU, Nanjing, Jiangsu Province, P. R. China.
... Association of larvae and adults of D. plicatus is supported by the similarity of partial COI haplotypes. Since the association of larvae and adults is not completely reliable based solely on comparisons of sequences of a single gene from one specimen each (e.g., Zhou et al. 2007), we analysed specimens from two different populations. At each locality some adult males of D. plicatus and unassigned larvae shared identical COI haplotypes (Table 2). ...
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A description of the larva of Drusus plicatus Radovanović is given for the first time. The most important diagnostic characters enabling separation from larvae of the other Drusinae from the southeast Europe are listed. Molecular, ecological, and ethological features and distribution patterns of the species are given. Additionally, information on the sympatric caddisfly species of the three springs where larvae and adults of D. plicatus were found and presented.
... Overall, intraspecific variability recorded in D. bosnicus (Table 1) is in line with the observed variability of the same COI fragment in populations of some other Drusus species (e.g., Pauls et al. 2009;Previšić et al. 2009), including species of the D. bosnicus Group (e.g., Kučinić et al. 2008). However, the association of larvae and adults are not completely reliable based solely on comparisons of sequences of a single gene from one specimen each (e.g., Zhou et al. 2007). In addition to the molecular genetic data, association of adults and larvae of D. bosnicus are further supported by: ...
In this study we present morphological, molecular and ecological features of the last instar larvae of Drusus bosnicus with data about distribution of this species in Bosnia and Herzegovina. We also included the most important diagnostic features enabling separation of larvae of D. bosnicus from larvae of the other European Drusinae and Trichoptera species.
... D. discolor (Pauls et al. 2006). As Zhou et al. (2007) pointed out, species boundaries based on genetic distance have to be defined specifically for each taxon. ...
Species of the genus Drusus usually inhabit cold springs and spring areas of mountain streams. Isolation of populations and a patchy distribution are reasons for high diversity and endemism in this genus. Nine taxa from the bosnicus group (genus Drusus) live in the Balkan Peninsula (Croatia, Bosnia and Herzegovina, Montenegro, Macedonia). This study has two main objectives. First, morphological, ecological, and genetic features (mitochondrial COI and 16S genes) of the final larval instar of Drusus radovanovici radovanovici from Bosnia and Herzegovina are analysed. Association of larvae and adults of D. radovanovici radovanovici is carried out according to genetic data (mitochondrial DNA) of adults and larvae, and supported by their distribution in Bosnia and Herzegovina. Second, phylogeny, taxonomy and distribution of taxa of the bosnicus group in the Balkan Peninsula are discussed. The analysis includes morphological and genetic data for the known larvae of the genus Drusus distributed in the Balkans. As a consequence, both subspecies of Drusus radovanovici are ranked as species (D. radovanovici and D. septentrionis stat. n.), and the status of other taxa from the bosnicus group is discussed. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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Adults and larvae of the genus Hydropsyche from Yun-nan and Si-chuan Provinces, China, were examined and the COI barcode gene sequences were obtained and analyzed, adults and larvae of 3 species were successfully associated. Of them, H. uvana Mey 1995 is a new record for the Chinese caddisfly fauna. The larvae of H. cerva Li & Tian 1990, H. penicillata Martynov 1931, and H. uvana Mey 1995 are described for the first time. The diagnostic features of the species are described and illustrated.
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North Carolina's Division of Environmental Management has a large number of semiquantitative stream macroinvertebrate collections that have been assigned water-quality ratings. These semiquantitative data use abundance values of Rare = 1, Common = 3, and Abundant = 10, allowing the calculation of mean abundance (range = 0-10) for each taxon across five water-quality classes. This information was used to derive tolerance values and classification criteria for a southeastern biotic index. Classification criteria were adjusted for both season and ecoregion, but no corrections were required for stream size. Tolerance values are listed for >500 North Carolina taxa, and are compared with a similar Wisconsin data base.
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Insect life stages are known imperfectly in many cases, and classifications are based often on only one or a few semaphoronts of a species. This is unfortunate as information in alternative life stages often is useful for scientific study. Although recent examples of DNA in taxonomy have emphasized the identification of indistinguishable species, such sequence data facilitate the association of life history stages and hold considerable promise in phylogenetic analysis, evolutionary studies, diagnostics, etc. These concepts are discussed here and an example is provided from diving beetles (Dytiscidae: Coleoptera). Three unknown larval specimens of an apparent species of Laccophilinae collected in Namibia were associated with the species Philodytes umbrinus (Motschulsky) using DNA sequence data. An 806-bp portion of the gene cytochrome oxidase I was sequenced from the unknown larvae. Several identified adult specimens of species of Laccophilinae from Namibia were also sequenced, including two P. umbrinus specimens and specimens from four Laccophilus Leach species. Additional species of Laccophilus from other areas of the world also were sequenced, as were specimens of Agabetes acuductus (Harris), Australphilus saltus Watts, Neptosternus boukali Hendrich & Balke and a species of Laccodytes Régimbart. Parsimony analysis resulted in two most parsimonious trees with the unknown larva unambiguously resolved in a group with both adult specimens of P. umbrinus (bootstrap value = 100%). The average pairwise p-distance between the unknown larva and adult P. umbrinus specimens averaged 0.09% (0–0.14%), compared with an average divergence between other conspecifics in the analysis of 0.24% (0–0.82%) and an overall average divergence between species of 13.49% (1.90–19.86%). Based on this, the unknown larvae were assigned to P. umbrinus. The larvae are diagnosed and described and their relationship with other Laccophilinae is discussed.
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The relationship between species is usually represented as a bifurcating tree with the branching points representing speciation events. The ancestry of genes taken from these species can also be represented as a tree, with the branching points representing ancestral genes. The time back to the branching points, and even the branching order, can be different between the two trees. This possibility is widely recognized, but the discrepancies are often thought to be small. A different picture is emerging from new empirical evidence, particularly that based on multiple loci or on surveys with a wide geographical scope. The discrepancies must be taken into account when estimating the timing of speciation events, especially the more recent branches. On the positive side, the different timings at different loci provide information about the ancestral populations.
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Sequence divergence in the second internal transcribed spacer (ITS2) of ribosomal DNA was examined for female specimens of Anopheles oswaldoi Peryassu from 7 localities in South America. The lengths of ITS2 for all mosquitoes ranged from 348 to 356 nucleotides. After alignment of these sequences, similarity ranged from 87 to 100%. Divergence was within the range of inter-specific differences for members of anopheline species complexes. Therefore, specimens were placed into 4 groups that may correspond to at least 4 cryptic species. One is probably related to An. oswaldoi sensu stricto and another to Anopheles konderi Galvão & Damasceno. The other 2 groups may correspond to species for which morphological identification remains to be clarified. These data provide evidence that An. oswaldoi comprise a complex of cryptic species and that DNA identification may help to resolve the taxonomic questions related to this group.
Sibling species A and B of Anopheles quadriannulatus (Theobald) are recognized as allopatric members of the Anopheles gambiae Giles complex of Afrotropical mosquitoes (Diptera: Culicidae). Species A represents An. quadriannulatus sensu stricto, widespread in southern Africa, whereas An. quadriannulatus species B occurs in Ethiopia. Because of difficulty of identification, distribution of An. quadriannulatus sensu lato remains poorly known. Cytotaxonomy and the standard DNA polymerase chain reaction (PCR) assay do not distinguish between species A and B of An. quadriannulatus. By optimizing the standard PCR assay (Scott et al., 1993) for identification of members of the An. gambiae complex, we identified two discriminant fragments of 153 bp and 900 bp from DNA of An. quadriannulatus species B, whereas only the 153 bp fragment was amplified for species A from South Africa. This modified PCR assay can therefore be used to distinguish between species A and B of An. quadriannulatus s.l. as well as other members of the An. gambiae complex.
Phylogeographic analyses have mostly been based on single-gene genealogies but it is unclear how conclusions from such studies depend on the choice of gene markers. We conducted a nested geographical clade analysis [A.R. Templeton, E. Routman and C.A. Phillips (1995) Genetics 140: 767-782] based on nuclear rDNA internal transcribed spacer region 2 (ITS2) sequences in the Timarcha goettingensis species complex (Coleoptera, Chrysomelidae), and compared the inferences with an updated version of previously published results using mitochondrial cytochrome oxidase II (COIl) sequences. Inferences from ITS2 suggest that patterns of marker distribution are mostly explained by restricted gene flow with isolation by distance. In contrast, COII revealed a history of geographical structure resulting from episodic population contiguous-range expansions. Both markers also show different genealogical patterns, which are associated to the effects of genetic introgression in a putative hybrid zone between two major lineages in the complex. Altogether, these differences are attributed to distinct population and/or evolutionary dynamics of the markers, and offer a more accurate phylogeographic description for the T. goettingensis complex.
'Living fossil' taxa, by definition, have no close relatives, and therefore no outgroup to provide a root to phylogenetic trees. We identify and use a molecular outgroup in the sole extant lineage of sphenodontid reptiles, which separated from other reptiles 230 million years ago. We isolated and sequenced a partial nuclear copy of the mitochondrial cytochrome b gene. We confirm the copy is indeed not mitochondrial, is older than all extant mitochondrial copies in Sphenodon (tuatara), and is therefore useful as a molecular outgroup. Under phylogenetic analysis, the nuclear copy places the root of the tuatara mitochondrial gene tree between the northern and the southern (Cook Strait) groups of islands of New Zealand that are the last refugia for Sphenodon. This analysis supports a previous mid-point rooted mitochondrial gene tree. The mitochondrial DNA tree conflicts with allozyme analyses which place a Cook Strait population equidistant to all northern and other Cook Strait populations. This population on North Brother Island is the only natural population of extant S. guntheri; thus, we suggest that the current species designations of tuatara require further investigation.
We analysed the secondary structure of two expansion segments (D2, D3) of the 28S rRNA gene from 229 leaf beetles (Coleoptera: Chrysomelidae), the majority of which are in the subfamily Galerucinae. The sequences were compared in a multiple sequence alignment, with secondary structure inferred primarily from the compensatory base changes in the conserved helices of the rRNA molecules. This comparative approach yielded thirty helices comprised of base pairs with positional covariation. Based on these leaf beetle sequences, we report an annotated secondary structural model for the D2 and D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related beetle taxa. This predicted structure, consisting of seven major compound helices, is mostly consistent with previously proposed models for the D2 and D3 expansion segments in insects. Despite a lack of conservation in the primary structure of these regions of insect 28S rRNA, the evolution of the secondary structure of these seven major motifs may be informative above the nucleotide level for higher-order phylogeny reconstruction of major insect lineages.