https://orcid.org/0000-0001-5614-7536
Figures
Abstract
Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.
Citation: Dvorak M, Dittmann IL, Pedrini-Martha V, Hamerlík L, Bitušík P, Stuchlik E, et al. (2024) Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria). PLoS ONE 19(2): e0298367. https://doi.org/10.1371/journal.pone.0298367
Editor: Miguel Cañedo-Argüelles Iglesias, Universitat de Barcelona, SPAIN
Received: September 7, 2023; Accepted: January 24, 2024; Published: February 15, 2024
Copyright: © 2024 Dvorak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This work was supported by the Austrian-Czech Lead Agency project—Austrian Science Fund FWF No. I 4568- B / Czech Science Foundation No. 20-00892L). I.L.D. is a recipient of a DOC Fellowship of the Austrian Academy of Sciences at the Department of Zoology at the University of Innsbruck. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The most striking effects of global climate change in the Alps include retreating glaciers [1] and an increasing temperature in mountain streams [2]. This process has far-reaching consequences, such as a shift in composition of the benthic fauna, where cold-adapted species are replaced by species from lower altitudes [3]. Tiny flies of the Chironomidae family (Diptera, Nematocera), informally known as chironomids, are one of the typical groups of aquatic insects that inhabit cold streams, including glacier-fed streams. Despite harsh conditions, cold streams in the European Alps host a dense but highly specialized invertebrate fauna, where chironomids are often the only group present [4, 5]. However, this only applies to specific taxa, as chironomids inhabit water ecosystems with a wide range of temperatures [6, 7].
Larvae of the Diamesinae subfamily, especially those of the Diamesa genus, are the most abundant chironomids of glacier-fed streams (kryal), even located by the glacier terminus; however, they can also dominate in cold streams not affected by meltwater (krenal, rhitral) [3, 8, 9]. Larval Diamesa are generally adapted to cold and well-oxygenated waters, springs, and occasionally to shallow stagnant waters and madicolous sites [10, 11]. Some Diamesa species live at high altitudes up to 5,600 m a.s.l. [12] and references therein and can complete their life cycles even when water temperature do not exceed 2°C [13, 14]. It is obvious that Diamesa species could play a key role in the assessment of changes in high-altitude streams and springs under climatic change [15]. However, effective monitoring is hampered by objective difficulties with the reliable identification of larval Diamesa.
In the Palaearctic Region, the genus comprises more than 100 species [16], 38 of which are known from Europe [17]. Reliable identification of the species is based on adult males [18] and to some extent on the pupae and pupal exuviae [19]. Even though modern identification keys are available for Diamesa larvae (e.g., [20, 21]), larval identification is a time-consuming and difficult process even for experienced taxonomists. Moreover, key structures, such as mentum and mandibles, may be worn or damaged, making identification even more difficult. In addition, some Diamesa species are indistinguishable as larvae. Thus, using up-to-date molecular methods along with morphological diagnosis is crucial for distinguishing similar species [22–24].
This study aims to identify Diamesa species from brooks in the Ötztal Alps, Austria, and compare the morphological identification of larvae with the identification based on molecular markers using DNA barcoding methods. The success of DNA barcoding relies on the availability of reference sequences for comparison. The COI gene has been widely adopted as a universal DNA barcode marker across various taxa [25–27]. The COI sequences are more commonly available in public databases, making it easier to match and identify unknown specimens.
The limitations of using COI alone for barcoding, such as similarities in sequences among closely related species and intraspecific variations [28–31], prompted consideration of suitable alternatives. Several studies have highlighted the potential of the internal transcribed spacer (ITS) region within ribosomal DNA for species identifications, including animals, plants, and fungi [32–35]. The ITS region, located in the nuclear genome, exhibits lower intraspecific variability than COI while maintaining adequate interspecific variability, making it a perfect marker for species delineation [36]. Despite the usefulness of the nuclear ITS region in increasing the resolution of species identification, there are reasons why using only ITS might not be the optimal approach [37]. The molecular evolution rates of mitochondrial and nuclear genes can differ. Mitochondrial genes, like COI, often evolve faster than nuclear genes [38]. This faster evolution can be advantageous for distinguishing closely related species. Depending solely on the slower-evolving ITS may lead to insufficient divergence for accurate species discrimination in certain cases. Relying solely on ITS might also result in a lack of consistency and comparability with existing DNA barcode databases. The combination of COI and ITS proved to be the most effective approach, allowing accurate sorting of species into distinct groups [36, 37]. Combining both mitochondrial and nuclear markers provides complementary information [37]. Mitochondrial markers are maternally inherited and can provide insights into historical relationships, while nuclear markers offer information on recent evolutionary events [39]. The combination of both markers enhances the overall resolution and accuracy of species delineation [36, 37].
One potential application of DNA barcoding for Diamesa species identification is in the monitoring of current environmental changes. As mentioned earlier, global warming and related environmental changes are causing the disappearance of glaciers in the Alps and an increase in the water temperature of mountain streams. We assume that chironomids of the Diamesa genus have a great bioindication potential and can be used to assess future changes in the quality of these vulnerable ecosystems.
Materials and methods
Sampling
Sampling sites are situated near Obergurgl (community of Sölden, Tyrol, Austria) in three neighbouring valleys–Rotmoostal, Königstal, and Timmelstal. The focus was on Rotmoostal, where a stream (Rotmoosache) is fed by meltwater from the Rotmoosferner and the Wasserfallferner. Rotmoos valley is part of the LTER site Obergurgl belonging to the LTSER platform Tyrolean Alps and to the national and international long-term ecological research networks (LTER-Austria, LTER Europe, and ILTER). In that stream, we expected the harshest conditions out of the three sampled streams and the presence of the Diamesa-dominated chironomid fauna. The other alpine streams in the Königstal and Timmelstal valleys (Königsbach and Timmelsbach) are spring-fed streams with no glaciers in their catchments. The sampling sites (five in the Rotmoostal, two in the Königstal, and one in the Timmelstal) were selected to cover a gradient of harshness (see [40], Fig 1, Table 1 for details). All sampling sites are situated above the treeline. The local bedrock is composed of granitic rocks with marble layers, feldspar, and mica schists. Chironomid larvae were sampled using a kick-sampling net (frame size of 25 × 17 cm, mesh size of 500 μm, sampling time approximately 5 minutes), stored in a thermos flask, and immediately transferred to a laboratory in the nearby University Center Obergurgl, Austria, and sorted under a dissecting microscope. Chironomids of the genus Diamesa were subsequently treated in a specific way (see below).
Sampling on August 22, 2020 (RM3, RM4), August 23, 2020 (RM0, RM1, RM2), and July 20, 2021 (TJ1, KT1, KT2). For sapling site codes see Table 1; Photos: D. Vondrák.
Abbreviations: GF–glacier-fed, NG–non-glacial.
Morphological identification
Each larva was dissected in a drop of distilled water to prevent drying and collapsing body structures. The head and the posterior part of the abdomen of each specimen were embedded in the Euparal microscopy mountant (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) on microscope slides for further taxonomic identification. The posterior part of the abdomen always included posterior parapodes, anal tubules, and anal setae, and the head was oriented by the ventral side up to show important morphological characters. Taxonomic identification followed Rossaro and Lencioni [20, 21]. Results of morphological identification are shown after sample ID in Fig 2. If individual species were morphologically indistinguishable, we tried to assign particular samples to respective clades and indicated that with the group abbreviation (gr.) in the sample name.
The first three letters in each individual’s code represent the sampling site code (see Table 1). The species names after the sample ID refer to the morphological identification, while colour coding for species is based on molecular assignments.
DNA extraction, PCR amplification and sequencing
A small part of the remaining body (up to 1 mm) was used for DNA extraction by the prepGem Universal kit (MicroGem; Southhampton, UK). Briefly, 44 μl of dd H2O DEPC, 5 μl of 10x blue buffer (prepGem Universal kit), and 1 μl of prepGEM were added to the sample. After 15 min at 75°C, the sample was heated up for 2 min to 95°C. After cooling, samples were stored at -20°C and were ready to be used for DNA barcoding.
PCR was performed with Advantage 2 Polymerase Mix (Takara, Japan). Primers sequences were outsourced from Folmer et al. [41] (LCO1490: 5’-ggtcaacaaatcataaagatattgg-3’, HC02198: 5’-taaacttcagggtgaccaaaaaatca-3’) with an expected product length of 658 base pairs. Briefly, a denaturation cycle of 95°C for 8 min was followed by 33 cycles with 95°C for 30 s, 57°C for 30 s, and 68°C for 1 min, with a final 10 min extension step at 70°C. PCR products for sequencing with Microsynth (Switzerland) were cleaned by QIAquick PCR Purification Kit (Qiagen, Germany). For quality control, amplified fragments were separated by size through electrophoresis on a 1.5% agarose gel in TEA buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA).
To improve resolution, we performed additional identification based on a section of nuclear ribosomal ITS. Primers were outsourced from Newburn & Krane [42] (18S: 5’-gatgttctgggcggcacgcg-3’, 28S: 5’-ttggtttcttttcctcccct-3’). PCR reactions of 25 μl were performed as follows: a denaturation cycle of 95°C for 8 min was followed by 30 cycles with 95°C for 30 s, 62.5°C for 30 s and 68°C for 1 min, with a final 10 min extension step at 70°C.
Phylogenetic analyses
Sequences for each gene were separately aligned with MAFFT G-INS-i v7.490 [43]. They were manually trimmed and concatenated to form a supermatrix (COI 1-453bp + ITS 454-3088bp). For phylogenetic reconstruction, we pursued maximum likelihood reconstructions using RAxML v8.2.10 [44]. The best model (GTR+G+I, same for both) was determined for individual alignments by MEGA software v11.0.13 [45]. The best ML tree was selected out of 1000 tree searches and 1000 separate bootstrap replicates were performed. Bootstrap support was mapped for each supermatrix onto the best ML tree. This was done both for the concatenated supermatrix (Fig 2) and for individual genes separately (S1 and S2 Figs). In the case of COI, we included known chironomid sequences from the database (S3 Table) to enhance the identification of species by clustering these known sequences with the clusters formed by our samples. This was crucial for the identification of morphologically indistinguishable larval samples, such as Diamesa modesta and Diamesa latitarsis. There were no records of Diamesa ITS sequences in GenBank at the time of this study (2023). Phylogenetic trees were visualised in Figtree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/) and redesigned in Adobe Illustrator CC (2014). The analysed sequences are available in the GenBank database under respective accession numbers (S2 Table). Further datasets from this study are available from the corresponding author upon request.
Results
In total, 129 specimens were analysed, with 101 sampled from the glacier-fed stream of Rotmoostal and the remaining 28 from the spring-fed streams in neighbouring valleys of Königstal and Timmelstal. This study focused on the subfamily Diamesinae, particularly the genus Diamesa (in total 104 specimens). However, several other taxa from the subfamilies Orthocladiinae and Chironominae were also analysed as outliers or accidentally in the case of small and dark Orthocladiinae taxa that resembled Diamesa larvae. Among the chironomid specimens collected from the study area, three Diamesa species were identified to occur only in the glacial-fed stream of Rotmoostal and were absent in the samples from the spring-fed streams of Königstal and Timmelstal (KT1, KT2, and TJ1). These exclusively glacial-fed stream species were Diamesa bertrami Edwards, 1935, D. modesta Serra-Tosio, 1967, and D. steinboecki Goetghebuer, 1933 (Table 2). Regarding the spring-fed streams, they were dominated by the subfamily Orthocladiinae and Chironominae. Particularly at the sampling site KT1, Diamesa species were completely absent in our sample set (Table 2).
The phylogenetic tree species delimitation analysis, illustrated in S1 and S2 Figs uses cytochrome oxidase subunit 1 (COI) marker gene sequences and internal transcribed spacer 1 and 2 (ITS) marker sequences. The COI tree (S1 Fig), incorporating known GenBank sequences as a reference, facilitated the identification of distinct species clusters. Notably, despite the morphologically indistinguishable species of Diamesa modesta and Diamesa latitarsis, these sequences formed separate clusters, with D. modesta aligning well with the known GenBank sequence (S3 Table). The second cluster did not align with the Diamesa latitarsis sequence from the GenBank (S3 Table); however, it was distinct from the cluster confidently identified as Diamesa modesta. Given the certainty of our morphological identification of larval samples as either modesta or latitarsis, we confidently classify the second cluster as Diamesa latitarsis. Discrepancies between the GenBank sequence and our sequences may stem from limitations inherent in the COI gene described earlier.
Nevertheless, the individual COI and ITS phylogenetic trees revealed a lack of adequate species resolution based on morphological analysis. To address this limitation and distinguish between Diamesa species, we employed concatenated sequences.
In the resulting phylogenetic tree (Fig 2), two clades of the Diamesa cluster were polyphyletic with a clade of Orthocladiinae. The main clade comprises Diamesa steinboecki, Diamesa bertrami, the members of the Diamesa cinerella group (likely D. cinerella Meigen, 1835 and D. starmachi Kownacki & Kownacka, 1970), and the so-called Diamesa latitarsis group (mainly D. modesta, D. latitarsis Goetghebuer, 1921, D. goetghebueri Pagast, 1947). The tree topology shows that the D. cinerella group builds a sister group to D. steinboecki, D. bertrami, as well as to the D. latitarsis group. In our tree, the D. latitarsis group mainly comprises sequences of D. goetghebueri, D. modesta and D. latitarsis. Besides a few exceptions, the representatives of the corresponding species appear monophyletic, forming three clades (Fig 2). The first branching is D. modesta; D. goetghebueri and D. latitarsis appear as sister clades. Only three sequences (RM3-004-Diamesa_latitarsis gr., TJ1-006-Diamesa_sp., and RM3-009-Diamesa_latitarsis_gr.; see Fig 2) of the D. latitarsis group cluster separately and do not associate with any of the three main clades formed by D. goetghebueri, D. modesta, or D. latitarsis.
Discussion
In recent years, the use of DNA barcoding in species identification has gained increasing popularity in many fields of biology. DNA barcoding is a technique that involves the use of a short DNA sequence to identify species. The DNA sequence used for barcoding is usually a fragment of a specific gene, such as the mitochondrial cytochrome oxidase subunit I (COI) gene. The COI gene is highly conserved within species but highly variable between species, making it an excellent tool for species identification. However, there are some limitations to the use of the COI gene as a barcode marker for species determination [22]. One of the limitations is that some closely related species may have very similar COI gene sequences, making it difficult to distinguish between them [22, 46]. In addition, some species may have intraspecific variations in their COI gene sequence, leading to potential misidentification [47]. Although established methods of DNA barcoding for chironomid species utilize fragments of the mitochondrial COI, it doesn’t provide sufficient resolution to distinguish all Diamesa species (S1 Fig).
To overcome these limitations and to increase the resolution of barcoding, we have been exploring the use of additional genetic markers for DNA barcoding. One such marker is the ITS region, which is located between the 18S and 26S rRNA genes in the nuclear genome. The ITS region has been found to have higher variability than the COI gene, making it a potentially useful marker for species delineation. The ITS is generally used for barcoding studies with animals and plants [32, 33, 35]. While the ITS alone improved the resolution of studied species, it exhibits some level of conservation within closely related species. In our case, this mainly concerns the Diamesa latitarsis group, where the individual species within this taxonomic cluster exhibited insufficient separation into distinct clusters (S2 Fig). For other clades, Diamesa bertrami, steinboecki, cinerella gr. and Orthocladine sp. separation was sufficient (S2 Fig). The most effective approach was a combination of COI and ITS together and a running analysis of the combined sequence of both. In Diamesa larvae, the combination of both markers allows for more accurate species delineation. This method enabled us to sort all analysed species into species groups. Our findings were compared with already published sequences from the NCBI database and the results of our taxonomical identification based on morphological features.
Identifying Diamesa larvae to species level is a difficult task due to the heavily sclerotized, frequently dark head, often worn mouthparts (mentum and mandibles), extreme morphological similarity of Diamesa species, and high variability of morphological features even within a species. This similarity is particularly evident among larvae of closely related species (i.e., members of a species group), which may share the same habitats and have similar ecological niches. The challenge of identifying Diamesa larvae to the species level is compounded by the fact that only larvae of about one-third of the described species are known [11], and there are quite few identification keys dealing with them (e.g., [20, 21, 48–51]); the precise identification of the genus is mainly based on the morphology of the adult males ([52]). Nevertheless, it is difficult to identify the larvae in the absence of adult males, and alternatively pupal exuviae or pharate adults. Additionally, the morphology of Diamesa larvae is subject to a high degree of plasticity in response to environmental factors, such as temperature, pH, and nutrient availability. This variability can make it difficult to distinguish between different species, as their morphological features may overlap or change in response to different environmental conditions [46]. Our tree shows that using the molecular marker gene COI in combination with ITS enables species identification even when traditional taxonomy reaches its limits.
Such an example is the differentiation between members of the so-called Diamesa latitarsis group [22, 46]. Besides the eponym species, D. latitarsis, this group includes six further species occurring in the Alps: D. goetghebueri, D. laticauda Serra-Tosio, 1964, D. lindrothi Goetghebuer, 1931, D. martae Kownacki & Kownacka, 1980 and D. modesta. These species were grouped based on the morphological features of the adult males [10, 53]. Montagna et al. [22] and Lencioni et al. [46] showed that this similarity of features likely has a shared common evolutionary origin, as this group appears monophyletic also in molecular phylogenetic analyses. In our study (Fig 2), the D. latitarsis group is mainly represented by D. goetghebueri, D. modesta and D. latitarsis which were distinguished based on the molecular data and form the three distinct monophyletic clades. At present, D. modesta is indistinguishable from D. latitarsis using morphological characters, and D. goetghebueri can only be distinguished in some larvae (see [20, 21]). We combined the results of our molecular analysis with COI alignment to known sequences of the NCBI database (S3 Table). With the help of our phylogenetic tree majority of these cases of potential misidentifications are easy to recognize and assign to the correct species (Fig 2). However, more difficult is the interpretation of the sequences clustering besides the main clades (RM3-004-Diamesa_latitarsis_gr., TJ1-006-Diamesa_sp., RM3-009-Diamesa_latitarsis_gr.; see Fig 2). Just with morphological characters and without further molecular references we were not able to identify the samples to the species level. As they do not cluster within the main clades in the tree, it might be possible that each of these sequences represents one of the remaining alpine species of the D. latitarsis group, i.e., D. laticauda, D. lindrothi or D. martae (larvae of the last two mentioned are unknown). However, we would need more data to clarify the phylogenetic affiliation of these sequences. However, the species mentioned above are not listed in the identification keys we used [20, 21] and consequently were not considered during morphological identification.
A similar problem arises in the clade D. cinerella group. In our phylogenetic tree, the D. cinerella group cluster does not seem monophyletic as D. starmachi clusters within. So far only two species from this group, D. cinerella and D. tonsa Haliday, 1856, were found in the Alps ([46] see Table 1 in their study). Taking this into account and based on the phylogenetic position of Diamesa starmachi in the tree, we assume the misidentification of the D. starmachi labelled sequences and a false signal of paraphyly inside the Diamesa cinerella group. Therefore, we hypothesise that the three affected sequences are not only D. starmachi but also D. cinerella or D. tonsa, leading to the monophyly of the Diamesa cinerella group. This hypothesis is also supported by the known distribution of D. starmachi, which occurs in the Carpathians in mountain and low mountain streams below 1200 m a.s.l. [54, 55], therefore its occurrence in the Rotmoosache is very unlikely. However, the paraphylie of D. cinerella or D. tonsa due to potential gene flow between these two species, as supposed by Montagna et al. [22], cannot be excluded. The previously performed molecular studies by Montagna et al. [22] and Lencioni et al. [46] also showed that these two species (D. cinerella and D. tonsa) cluster closely intertwined. It is hypothesised that they might represent different populations of the same species [46]. The morphological identification of larvae of these two species, entirely based on the head’s colour pattern, is quite difficult or impossible and is prone to misidentification. This is stressing again the importance of collaboration between taxonomists and molecular biologists, as well as the choice of suitable marker genes. In our study, we were not able to differentiate between the larvae of these two species either. Despite the usefulness of DNA barcoding in identifying Diamesa larvae at the species level, there are still limitations to this approach. For example, the success of DNA barcoding relies on the availability of reference sequences for comparison. If there are no reference sequences available for a particular species, it can be difficult to identify that species using DNA barcoding. So far, our study is the first to use the ITS marker gene in chironomids, making a comparison with data resulting from other studies challenging. However, it is noticeable that our tree shows two separate main clades in the Diamesa cinerella group (Fig 2). Further studies, maybe including adult males, would be necessary to see if these two clades represent D. cinerella and D. tonsa as two species, or if they belong to the same species as supposed by [46].
Another interesting aspect is the phylogenetic position of D. steinboecki. In our phylogenetic tree D. steinboecki, a clade including all remaining sequences of Diamesa and Orthocladiinae cluster polyphyletic. However, we do not doubt the monophyly of the genus Diamesa, due to this seeming polyphyly. Based on morphological characteristics, D. steinboecki was supposed to feature the ancestral type of this genus [56] and is shown to have an early branching position in phylogenetic trees (see [46]). We hypothesise this position is also supported by our dataset; however, it’s just not resolved by our tree (Fig 2). Regardless of its general position in the tree, this clade forms a monophyly and can be, supported by the morphological data, clearly identified as D. steinboecki. Also, the morphological and molecular determination of D. bertrami in our tree is distinct (Fig 2).
In summary, while the ITS region can be a valuable marker for DNA barcoding, using a combination of both COI and ITS markers is often preferred to leverage their respective strengths and overcome their individual limitations. This approach ensures a more robust and reliable identification of species in diverse biological studies. Our study shows that DNA barcoding combining COI and ITS data can improve the morphological identification of Diamesa species in alpine streams.
As mentioned in the introduction, these harsh environments often host only a few well-adapted species [57, 58] that can withstand these conditions. Some of the Diamesa species seem to be obligate inhabitants of glacier-fed streams; however, other Diamesa species may inhabit a wider range of running waters, including lowland streams and rivers (e.g., [2, 55, 59, 60). We observed D. steinboecki, D. modesta, and D. bertrami only in the glacial-fed Rotmoosache stream (see Table 2), which is in agreement with the reported higher affinity of those species for a given habitat type than in the case of D. latitarsis, D. goetghebueri, and members of the D. cinerella group (e.g., [2]). We did not record any representatives of the Diamesa genus at the sampling site KT1. It is plausible that this site is a periodic stream that dries up in late summer and freezes in winter and thus does not provide a suitable habitat for Diamesa species.
In general, larvae of most Diamesa species dwell in cold water of glacial or high mountain streams. Thus, the effect of glacial retreat on Diamesa species will be the isolation of their populations and the potential extinction of the most cold-restricted species due to constrained habitat preferences and limited migration abilities [52]. Even though difficulties in species identification of Diamesa larvae often hamper detailed knowledge of their ecology, it is obvious that some species are more cold-restricted and thus more prone to extinction than others. In the Southern Alps, for example, Diamesa steinboecki, D. latitarsis, D. modesta, D. goetghebueri and D. laticauda are known to be restricted to glacial habitats [15] and thus exposed to higher risks of extinction due to the progressive shrinkage of their habitats. In addition, rare species with restricted distributions can be heavily impacted by the glacier retreat and warming water. In the Tatra Mountains, where there are no recent glaciers, populations of some Diamesa species (D. steinboecki, D. latitarsis, D. nowickiana) survive in a few extremely cold streams and are considered critically endangered [61].
The advances in innovative identification methods using molecular markers may overcome the issues of morphological identification and increase the knowledge of the ecology and environmental limitations of different Diamesa species.
Future research should also aim to apply barcoding to other chironomid life stages, such as pupae. Although pupae (and pupal exuviae) provide more reliable diagnostic features than larvae, their identification is still uncertain, and the position of some species is tentative [19].
Conclusion
The use of DNA barcoding for species identification has numerous potential applications in ecology and conservation biology, including the monitoring of biodiversity, identification of invasive species, and assessment of the impacts of climate change on ecosystems. In the case of Diamesa larvae, which are important bioindicators in alpine streams, the combination of COI and ITS markers enabled a higher resolution in species identification compared to using either marker alone. This approach, supported by the utilization of morphological characters in larvae, allows for more precise species identification and the construction of distance-based phylogenetic trees for the genus Diamesa. Moreover, this more detailed identification within the genus can offer significant insights into the ecology of alpine streams, comprehension of which is becoming increasingly crucial in the context of the current climate changes.
Supporting information
S1 Table. Physico-chemical water parameters of studied sampling sites.
https://doi.org/10.1371/journal.pone.0298367.s001
(PDF)
S2 Table. GenBank accession numbers of analysed sequences.
GenBank database accession numbers of analysed sequences of cytochrome oxidase subunit 1 gene (COI) and ribosomal RNA gene including internal transcribed spacer 1 and 2 (ITS).
https://doi.org/10.1371/journal.pone.0298367.s002
(PDF)
S3 Table. GenBank accession numbers of known sequences.
GenBank database accession numbers of used sequences of cytochrome oxidase subunit 1 gene (COI) and ribosomal RNA gene including internal transcribed spacer 1 and 2 (ITS).
https://doi.org/10.1371/journal.pone.0298367.s003
(PDF)
S1 Fig. COI phylogenetic tree.
Phylogenetic tree showing species delimitation analysis based on the cytochrome oxidase subunit 1 (COI) marker gene sequences. The first three letters in each individual’s code represent the sampling site code (see Table 1).
https://doi.org/10.1371/journal.pone.0298367.s004
(TIF)
S2 Fig. ITS phylogenetic tree.
Phylogenetic tree showing species delimitation analysis based on the internal transcribed spacer 1 and 2 (ITS) marker sequences. The first three letters in each individual’s code represent the sampling site code (see Table 1).
https://doi.org/10.1371/journal.pone.0298367.s005
(TIF)
References
- 1. Sommer C, Malz P, Seehaus TC, Lippl S, Zemp M, Braun MH. Rapid glacier retreat and downwasting throughout the European Alps in the early 21st century. Nature Communications. 2020; 11: 3209. pmid:32587270
- 2. Lencioni V, Stella E, Zanoni MG, Bellin A. On the delay between water temperature and invertebrate community response to warming climate. Science of the Total Environment. 2022; 837: 155759. pmid:35533868
- 3. Lencioni V, Rossaro B. Microdistribution of chironomids (Diptera: Chironomidae) in Alpine streams: an autoecological perspective. Hydrobiologia. 2005; 533: 61–76.
- 4. Lencioni V, Maiolini B, Rossaro B. The kryal and rhithral chironomid community in the Care`Alto system (Italian central-eastern Alps). Verhandlungen der Internationale Vereinigung für Theoretische und Angewandte Limnologie. 2001; 27: 711–715.
- 5. Lods-Crozet B, Lencioni V, Ólafsson JS, Snook DL, Velle G, Brittain JE, et al. Chironomid (Diptera: Chironomidae) communities in six European glacier-fed streams. Freshwater Biology. 2001; 46: 1791–1809.
- 6. Rossaro B. Chironomids and temperature. Aquatic Insects. 1991; 13: 87–98.
- 7. Eggermont H., Heiri O. The chironomid-temperature relationship: expression in nature and palaeoenvironmental implications. Biological Reviews. 2012; 87, 430–456. pmid:22032243
- 8. Ward JV. Ecology of alpine streams. Freshwater Biology. 1994; 32: 277–294.
- 9.
Füreder L. High alpine streams: cold habitats for insect larvae. In: Margesin R, Schinner F (eds): Cold-Adapted Organisms. Springer, Berlin, Heidelberg. 1999. https://doi.org/10.1007/978-3-662-06285-2_10
- 10. Serra-Tosio B. Ecologìe et biogéographie des Diamesini d’Europe (Diptera, Chironomidae). Travaux Du Laboratorie D’ Hydrobiologie Et De Pisciculture De L’université De Grenoble. 1973; 63: 5–175.
- 11.
Sæther OA, Andersen T. 7. The larvae of Diamesinae (Diptera: Chironomidae) of the Holarctic Region–Keys and diagnoses. In: Andersen T, Sæther OA, Cranston PS, Epler JH (eds.): Chironomidae of the Holarctic Region. Keys and diagnoses. Larvae. Insect Systematics & Evolution. 2013; Supplement 66: 145–183.
- 12. Ferrington LC Jr. Global diversity of non-biting midges (Chironomidae; Insecta-Diptera) in freshwater. Hydrobiologia. 2008; 595: 447–455.
- 13. Burgherr P, Ward JV. Longitudinal and seasonal distribution patterns of the benthic fauna of an alpine glacial stream (Val Roseg, Swiss Alps). Freshwater Biology. 2001; 46: 1705–1721.
- 14. Milner AM, Brittain JE, Castella E, Petts GE. Trends of macroinvertebrate community structure in glacier-fed rivers in relation to environmental conditions: a synthesis. Freshwater Biology. 2001; 46(12): 1833–1847.
- 15. Rossaro B, Montagna M, Lencioni V. Environmental traits affect chironomid communities in glacial areas of the Southern Alps: evidence from a long‐lasting case study. Insect Conservation and Diversity. 2016; 9(3): 192–201.
- 16.
Ashe P, O’Connor JP. A World Catalogue of Chironomidae (Diptera). Part 1. Buchonomyiinae, Chilenomyiinae, Podonominae, Aphroteniinae, Tanypodinae, Usambaromyiinae, Diamesinae, Prodiamesinae and Telmatogetoninae. Irish Biogeographical Society & National Museum of Ireland, Dublin. 2009; 445. ISBN: 9780955080647.
- 17.
Spies M, Sæther OA. Chironomidae. Fauna Europaea: In: Beuk P, Pape T (Eds): Fauna Europaea: Diptera, Nematocera. Fauna Europaea version 2.6. 2013; Internet database: http://www.faunaeur.org (Accessed on 2023-06-29).
- 18.
Langton PH, Pinder LCV. Keys to the adult male Chironomidae of Britain and Ireland. 2 Vols. Freshwater Biological Association, Scientific publication. 2007; 64: 1–239 + 1–168.
- 19.
Langton PH, Visser H. Chironomidae exuviae. A key to pupal exuviae of the West Palaearctic Region. Interactive Identification System for the Europaean Limnofauna (IISEL), World Biodiversity Database, CD–ROM Series. 2003.
- 20. Rossaro B, Lencioni V. A key to larvae of Diamesa Meigen, 1835 (Diptera: Chironomidae), well known as adult males and pupae from Alps (Europe). Journal of Entomological and Acarological Research. 2015a; 47: 123–138.
- 21. Rossaro B, Lencioni V. A key to larvae of species belonging to the genus Diamesa from Alps and Apennines (Italy). European Journal of Environmental Sciences. 2015b; 5(1): 62–79.
- 22. Montagna M, Mereghetti V, Lencioni V, Rossaro B. Integrated Taxonomy and DNA Barcoding of Alpine Midges (Diptera: Chironomidae). PLoS ONE. 2016a; 11(3): e0149673. pmid:26938660
- 23. Krasheninnikov AB, Makarchenko EA, Semenchenko AA, Gavrilo MV, Vshivkova KA. Morphological description and DNA barcoding of some Diamesinae (Diptera, Chironomidae) from the Severnaya Zemlya Archipelago and the Vaigach Island (Russian Arctic). Zootaxa. 2020; 4802(3): 587–600. pmid:33056053
- 24. Makarchenko EA, Semenchenko AA, Palatov DM. Fauna and taxonomy of Diamesinae (Diptera, Chironomidae) from the Caucasus, with a morphological description and DNA barcoding of new taxa and a discussion of diagnostic problems for Diamesa Meigen and Pseudodiamesa Goetghebuer. Zootaxa. 2023; 5271(2): 313–328. pmid:37518126
- 25. Anslan S, Tedersoo L. Performance of cytochrome c oxidase subunit I (COI), ribosomal DNA Large Subunit (LSU) and Internal Transcribed Spacer 2 (ITS2) in DNA barcoding of Collembola. European Journal of Soil Biology. 2015; 69: 1–7.
- 26. Hebert PDN, Ratnasingham S, de Waard JR. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society B. 2003; 270: 96–99. pmid:12952648
- 27. Pentinsaari M, Salmela H, Mutanen M, Roslin T. Molecular evolution of a widely-adopted taxonomic marker (COI) across the animal tree of life. Scientific Reports. 2016; 6: 35275. pmid:27734964
- 28. Ebong MAS, Petit E, Le Gall P, Chen PP, Nieser N, Guilbert E, et al. Molecular Species Delimitation and Morphology of Aquatic and Sub-Aquatic Bugs (Heteroptera) in Cameroon. PLoS ONE. 2016; 11(5): e0154905. pmid:27149077
- 29. Laurito M, de Oliveira TMP, Almiron WR, Sallum MAM. COI barcode versus morphological identification of Culex (Culex) (Diptera: Culicidae) species: a case study using samples from Argentina and Brazil. Memorias do Instituto Oswaldo Cruz. 2013; 108. pmid:24473810
- 30. Park DS, Foottit R, Maw E, Hebert PDN. Barcoding Bugs: DNA-Based Identification of the True Bugs (Insecta: Hemiptera: Heteroptera). PLoS ONE. 2011; 6(4): e18749. pmid:21526211
- 31. Wang G, Li C, Guo X, Xing D, Dong Y, Wang Z, et al. Identifying the Main Mosquito Species in China Based on DNA Barcoding. PLoS ONE. 2012; 7(10): e47051. pmid:23071708
- 32. Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS, Donoghue MJ. The ITS Region of Nuclear Ribosomal DNA: A Valuable Source of Evidence on Angiosperm Phylogeny. Annals of the Missouri Botanical Garden. 1995; 82(2):247–277.
- 33. Chen S, Yao H, Han J, Liu C, Song J, Shi L, et al. Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. PLoS ONE. 2010; 5(1): e8613. pmid:20062805
- 34. Schoch CL, Seifert KA, Huhndorf S, Schindel D. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences. 2012; 109(16). pmid:22454494
- 35. Yao H, Song J, Liu C, Luo K, Han J, Li Y, et al. Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals. PLoS ONE. 2010; 5(10): e13102. pmid:20957043
- 36.
Rajasegaran P, Koosakulnirand S, Tan KK, Khoo JJ, Suliman Y, Mansor MS, et al. Multi-locus sequence analysis indicates potential cryptic speciation in the chigger mite Neoschoengastia gallinarum (Hatori, 1920) parasitizing birds in Asia. 2023; PREPRINT available at Research Square. https://doi.org/10.21203/rs.3.rs-3682938/v1
- 37. Bourke BP, Oliveira TP, Suesdek L, Bergo ES, Sallum MAM. A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae). Parasites Vectors. 2013; 6: 11. pmid:23597081
- 38. Brown WM, George M Jr, Wilson AC. Rapid evolution of animal mitochondrial DNA. Proceedings of the National Academy of Sciences of the United States of America. 1979; 76(4): 1967–1971. pmid:109836
- 39. Schon EA, DiMauro S, Hirano M. Human mitochondrial DNA: roles of inherited and somatic mutations. Nature Reviews Genetics. 2012; 13(12): 878–90. pmid:23154810
- 40. Dvorak M, Dittmann IL, Pedrini-Martha V, Hamerlík L, Bitušík P, Stuchlik E, et al. Energy status of chironomid larvae (Diptera: Chironomidae) from high alpine rivers (Tyrol, Austria). Comparative Biochemistry and Physiology, Part A. 2023; 284: 111477. pmid:37419411
- 41. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology. 1994; 3(5): 294–299. pmid:7881515.
- 42.
Newburn E, Krane D. Molecular Identification of Chironomid Species. In: Lipnick RL, Mason RP, Phillips ML, Pittman CU Jr (Eds): Chemicals in the Environment. American chemical society, Washington DC, USA. 2002: 363–382. https://doi.org/10.1021/bk-2002-0806.ch021
- 43. Katoh K, Standley DM. MAFFT Multiple sequence alignment software version 7: Improvements in performance and usability. Molecular biology and evolution. 2013; 30(4): 772–780. pmid:23329690
- 44. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–1313. pmid:24451623
- 45. Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis version 11. Molecular biology and evolution. 2021; 38(7): 3022–3027. pmid:33892491
- 46. Lencioni V, Rodriguez-Prieto A, Allegrucci G. Congruence between molecular and morphological systematics of Alpine non-biting midges (Chironomidae, Diamesinae). Zoologica Scripta. 2021; 50: 455–472.
- 47. Zhang H, Bu W. Exploring Large-Scale Patterns of Genetic Variation in the COI Gene among Insecta: Implications for DNA Barcoding and Threshold-Based Species Delimitation Studies. Insects. 2022; 13(5): 425–436. pmid:35621761
- 48. Pankratova VY. Key to the larvae of the genera of the sub-family Orthocladiinae from Larvae and Pupae of midges of the sub-family Orthocladiinae of the fauna of the USSR. FBA Translations (New Series). 1970; 54: 51–5.
- 49. Rossaro B. Description of some unknown larvae of Diamesa genus and corrections to previous descriptions (Diptera, Chrironomidae). Archiv für Hydrobiologie. 1980; 90(3): 298–308. ISSN 0003-9136.
- 50.
Ferrarese U, Rossaro B. Chironomidi 12., 1 (Diptera, Chironomidae: generalià, Diamesinae, Prodiamesinae). Guide per il riconoscimento delle specie animali delle acque interne italiane C.N.R. AQ/1/129. 1981; 12: 1–97.
- 51. Schmid PE. A key to the Chironomidae and their instars from Austrian Danube region streams and rivers. Part I. Diamesinae Prodiamesinae and Orthocladiinae. 1993.
- 52. Montagna M, Urbanelli S, Rossaro B. The species of the genus Diamesa (Diptera, Chironomidae) known to occur in Italian Alps and Apennines. Zootaxa. 2016b; 4193(2): 317–331. pmid:27988720
- 53.
Kownacki A, Kownacka M. Diamesa martae sp. n. (Diptera: Chironomidae) and zoogerographical distribution of the species of Diamesa latitarsis group. Acta Hydrobiologica. 1980; 22/3: 313–326.
- 54.
Kownacki A, Kownacka M. Diamesa starmachi sp. n. (Diptera, Chironomidae). Bulletin de l´Academie Polonaise des Sciences. 1970; 18/12: 777–780.
- 55. Bitušík P, Svitok M, Dragúňová M. The actual longitudinal zonation of the river Hron (Slovakia) based on chironomid assemblages (Diptera, Chironomidae). Acta Universitatis Carolinae Biologica. 2006; 50: 5–17.
- 56.
Kownacki A. Ecology and biogeography of the Diamesa steinboecki group. In: Lellák J (ed): Proceedings of the 6th International Symposium Chironomidae, Prague, 17–20 August 1976. Acta Universitatis Carolinae. Biologica. 1978: 95–102.
- 57. Lencioni V. Survival strategies of freshwater insects in cold environments. Journal of limnology. 2004; 63(1s): 45–55.
- 58. Khamis K, Hannah DM, Brown LE, Tiberti R, Milner AM. The use of invertebrates as indicators of environmental change in alpine rivers and lakes. Science of the Total Environment. 2014; 493: 1242–1254. pmid:24650750
- 59. Bitušík P. A biological assessment of the Turiec river and its selected tributaries using chironomid pupal exuviae (Diptera, Chironomidae). Dipterologica bohemoslovaca. 1995; 7: 17–26.
- 60. Hroch M, Brabec K. Analysis of multiple-pressure pattern in rivers and its effects on the structure of macroinvertebrate communities. Limnologica. 2022; 97: 126027.
- 61. Kownacki A. Znaczenie i ochrona Chironomidae (Diptera, Insecta) w ekosystemach wodnych Polski. Forum Faunistyczne. 2011; 1(1): 4–11).