4.1 Macroinvertebrate identification based on DNA
barcoding
The species identification using DNA
barcoding is based on the fact that the genetic distance between two
species is much greater than that within a species. It has been proposed
to take 2% as the threshold value of species delimitation and, in
general, the average genetic distance between two species is over 10
times of that within a species (Hebert et al., 2003; Ward et al., 2009).
In this study, the average of interspecific K2P distance (16.37%) was
21-fold higher than that of intraspecific K2P distance (0.78%), which
meets the criteria of threshold value that the average of interspecific
genetic distance is 10 more times than that of intraspecific genetic
distance. The distribution histogram of intraspecific and interspecific
distances shows that 85.19% and 97.04% of the intraspecific distances
were less than 1% and 2%, respectively, and 97.88% of the
interspecific distances were greater than 6%, implying very little
overlap between intraspecific and interspecific genetic distances. Based
on the Barcode Gap analysis, the minimum interspecific distances to the
nearest neighbor were larger than the maximum intraspecific distance for
187 species (98.9% of all species). Only for two species (P.
laetum and P. bullum ), the maximum intraspecific distances
overlapped with the NN distance, leading to the absence of a barcode
gap. These results reveal that DNA barcoding based on COI gene is an
effective method for the species identification of benthic
macroinvertebrates in the transboundary rivers of northwest China.
Based on the NJ method, ABGD method and BIN analysis, high levels of
genetic distance and genetic lineages were observed in nine taxa
(Radix auricularia, Epeorus sp5, Rhithrogena tianshanica,
Ameletus montanus, Atherix sp. XJ, Glyptotendipes sp. XJ,
Euryhapsis sp, Dicranota guerini and Cricotopus ornatus ),
suggesting the presence of cryptic species diversity of benthic
macroinvertebrates in these transboundary rivers of northwest China.
Although Hebert and Ward proposed the threshold value (2%) of species
differentiation based on DNA barcoding
(Hebert et al., 2003; Ward et
al., 2009), the differences in genetic differentiation can occur in
different geographical populations for the same species, and thus the
genetic distance can exceed the threshold value of 2% for species
classification (Tajiama et al., 1983; Hickerson et al., 2006; Wardet
al., 2009). In present study, the nine species exhibited high
intraspecific genetic distance and multiple genetic lineages, and this
was consistent with the conclusions of Ward et al. (2009). Meanwhile,
our results support the conclusion that the genetic distance between
different geographical populations of the conspecifics can exceed 2%
(Hebert et al., 2003b; Ward et al., 2005). Coincidentally, the two or
three respective molecular lineages/clusters observed in R.
auricularia , Glyptotendipes sp. XJ, C. ornatus , D.
guerini , Atherix sp. XJ, R. tianshanica , Epeorussp5 and A. montanus corresponded to different geographical areas,
implying that biogeographic events result in a great intraspecific
divergence for these species. And geographical distance can play an
important role in the formation of high intraspecific genetic distance
or cryptic species.
Likewise, the genetic differentiation within one species occurred at
different sample sites or geographic scales for the Irtysh river, Emin
river and Ili river. For instance, the sampling site of Bieliezeke in
the Irtysh river is nearly 700 km away from that of Qiaoerma in the Ili
river. However, for species Ameletus montanus , four individuals
in site Bieliezeke showed high intraspecific divergence (up to 15.07%)
with 23 individuals in site Qiaoerma, suggesting that the divergence
reaches an interspecific level. Moreover, the BIN and ABGD analysis
divided them into two different groups, and the NJ tree analysis also
formed two main branches. Through rechecking the specimens, we did not
find any morphological feature that represents different species. In
contrast, the altitude (2294 m) of site Qiaoerma is higher than that of
site Bieliezeke (640 m). And the habitats of these two sites are totally
different as well as the nuptial flight, breeding time of the two
populations. This can provide the two populations with ideal criteria
for cryptic species due to long evolution time and the difference in the
habitats. Likewise, the species D. guerini showed high
intraspecific divergence (up to 7.26%) between the Irtysh River and Ili
River populations. Even in the same river (Ili River), the speciesR. auricularia yielded an intraspecific divergence of 4.3%
between site Nileke and site Zhaosu, that are separated by a distance of
150 km. Specifically, these two sites are situated at two different
tributaries in the upstream of Ili River and isolated by the Wusun
Mountain. In present study, DNA barcoding proves to be effective for the
species identification of benthic macroinvertebrates in most cases.
However, as a preliminary hypothesis of species classification, DNA
barcodes can be supplemented by morphological, ecological nuclear DNA
and other non-molecular data in what respects the existence of cryptic
species.
DNA barcoding has been widely used for species identification (Andrea et
al., 2016; Versteirt et al., 2015). However, whether DNA barcoding can
distinguish the individuals from different geographical populations,
subspecies or biotype, remains unknow. The NJ tree showed that the
conspecifics of barcoding sequences in the present study first clustered
together, and then clustered with those of other areas (Germany, United
States, Mexico, Canada, Norway, Italy, Finland etc.). In the NJ tree,
both S. striata and C. pallidivittatus covered two
subclusters, which were in accordance with the sampling locations. The
same geographical populations clustered together with high support
values, and the phylogenetic tree indicated that the evolution of
geographical population was related to geographical distance. As a
result, we inferred that the population differentiation of benthic
macroinvertebrates in these four rivers was ascribed to geographical
isolation. It has been reported that COI genes are not sensitive enough
to identify intraspecific variation, especially when the geographical
differentiation of populations is not long enough to form a single
pattern (Verheyen et al., 2003; Aliabadian and Kaboli., 2008). This
phenomenon was also observed in our study. In terms of the genetic
structure analysis among different geographic population, common
haplotypes existed in three adjacent geographic population of three
mayfly species, whereas different geographical populations generated a
certain degree of gene flow, intra-population and inter-population
genetic divergence. In the NJ tree, the geographical populations did not
divide into different branches according to different geographical
locations. The lacking of genetic differentiation among populations made
COI gene unable to effectively distinguish infraspecific category.
However, if the influence of geographical isolation and ecological
environment lead to the accumulation of genetic differentiation among
populations, DNA barcoding have the potential to distinguish the
geographical populations, subspecies or biotype (Monaghan et al., 2006).
Although COI gene has great potential of species identification at a
species level, but for infraspecific identification, the evolution rate
of COI genes can be limited due to a protein-coding gene. Therefore, COI
gene is not sensitive enough to identify populations with tiny genetic
differentiation, in which the geographical locations are adjacent and
the formation of geographical isolation pattern is not long enough. In
this case, more factors (e.g., increasing the length of DNA barcoding)
should be considered, especially for those non-protein-coding genes with
faster evolution rate.
4.2 Environmental
and biodiversity assessment
Phylogenetic diversity (PD) represents the adaptive potential of a
community in that higher genetic diversity means greater adaptability to
environmental changes (Vellend et al., 2011). In addition, PD indices
measure the evolutionary relationship between species, and can reflect
different aspects of biodiversity that are not captured by traditional
indices based on taxonomic diversity (e.g., Petchey & Gaston, 2002;
Cianciaruso et al., 2009; Vellend et al., 2011; Weiher, 2011).
Therefore, PD metrics have the great potential to be used in
biomonitoring of aquatic ecosystems (Vandewalle et al., 2010).
Our results demonstrated that human disturbance led to a decrease of PD
since values at reference sites were significantly higher than those in
disturbed sites (Fig. 5). Lower PD values in disturbance sites suggest
that human activities impact not only the taxonomic diversity of rivers,
but also the evolutionary history shared by the component species. The
PD metrics responded to environmental impact and complemented the
information provided by classical metrics. This suggests that PD metrics
can reflect a certain environmental stress and thus can be used as a
metrics of Index of Biotic Integrity (IBI) to reflect the degree of
disturbance in river systems.
We evaluated the water quality of the Irtysh River and Ili River based
on the combination of macroinvertebrate assemblages and
biotic index. Moreover, PD was
included into the candidate parameters of Index of Biotic Integrity
(IBI). The results of water quality assessment in the Irtysh River
through chemical measurements were identical with those of Liu et al.
(2002). Thus, the proposed IBI system is appropriate to the studied
rivers, and can serve as an effective measure of environment monitoring
in river systems. In summary, DNA
barcoding can provide a quantitative method to differentiate between
Good and Bad water quality. Moreover, our study showed that species
identifications based on DNA barcoding have the potential to detect tiny
changes in stream condition (e.g., taxa abundance, cryptic species and
multiple species lineages).
In conclusion, for the first time, our study constructed a DNA barcoding
reference library of benthic macroinvertebrates in transboundary rivers
of northwest China, which provides a coverage for 1227 sequences, 189
species, nine taxa of macroinvertebrates. The integration of Barcode Gap
analysis, Tree-based methods, ABGD analysis and BIN analysis was adopted
to compare with those by morphological identification. Our results
demonstrated that DNA barcoding based on COI is an effective method to
clarify species boundaries and quantitatively evaluate species
diversity, which can be used to evaluate lineages diversity and
phylogenetic structure, as well as
assess biodiversity and environmental condition for specific areas.