A well-established invasive Sinanodonta woodiana population was discovered in a floodplain lake of the upstream section of the Irrawaddy River basin, Kachin State, Myanmar. The DNA barcoding reveals that the population belongs to the temperate invasive mtDNA lineage and represents the same cytochrome c oxidase subunit I haplotype, which has been recorded in the invasive European populations. It is the most southern location of a population appertaining to this highly invasive haplotype known to date. The actual distribution of this alien species in Myanmar is still unknown, although it appears to be rather not widespread. Its possible dispersal through the country may affect native benthos communities, which include many unique endemic taxa. However, further expansion of the temperate lineage across South East Asia is supposed to be limited due to a specific environment of tropical floodplain freshwater systems that appears too warm for this cryptic taxon.
The Chinese pond mussel, Sinanodonta woodiana (Lea, 1834) is a well-known invasive species of the Unionidae, which is widely spread almost around the world together with its alien fish hosts (Douda, Vrtílek, Slavík, & Reichard, 2012; Lajtner & Crnčan, 2011; Lopes-Lima et al., 2017; Watters, 1997). Recent molecular studies reveal that S. woodiana is rather a complex of several closely related species because it comprises at least seven deeply divergent mtDNA lineages (Bolotov et al., 2016). Among them, the two lineages could only be considered as invasive. The first one is the tropical invasive lineage, which is currently widespread across the Malay Peninsula, the Philippines, and through Indonesia to the Lesser Sunda Islands (Bolotov et al., 2016; Zieritz et al., 2016). The origin of this successful invader is uncertain, but several authors placed it within Taiwan or the southern regions of continental China based on the analysis of primary sources for introduced fishes (Bolotov et al., 2016; Djajasasmita, 1982; Watters, 1997). The second one is the temperate invasive lineage, which has broad nonnative range in Europe, but most likely originated from the Yangtze drainage basin (Bolotov et al., 2016; Watters, 1997).
S. woodiana may affect native mussel populations, including the negative impact via cross-resistance of host fishes (Donrovich et al., 2017; Sousa, Novais, Costa, & Strayer, 2014). Adult Sinanodonta mussels can compete effectively with indigenous taxa for space, food, and host fishes. Their ability to modify natural ecosystems influences biological, physical, and chemical parameters of water environment (Bolotov et al., 2016; Douda et al., 2012; Guarneri et al., 2014; Sousa, Gutiérrez & Aldridge, 2009, 2014; Watters, 1997). An infection of fishes with the glochidia of Sinanodonta may negatively affect their physiological conditions, size, and weight (Douda et al., 2017).
In the present correspondence, we report the first occurrence of S. woodiana from Myanmar. In addition, we discuss the possibility of the further spread of this lineage of S. woodiana species complex across river systems of Southeast Asia and its possible effects on the native freshwater communities.
Available cytochrome c oxidase subunit I (COI) sequences of S. woodiana species complex and its sister taxa were downloaded from the BOLD IDS and NCBI’s GenBank, resulting in 68 sequences from Europe, China, Russia, South Korea, Japan, Malaysia, the Philippines, and Indonesia (Table 1). The COI sequence, which was provided for the S. aff. woodiana specimen from Romania (NCBI’s GenBank acc. no. JQ435822), was not included (see Bolotov et al., 2016). We have analyzed three available specimens of S. woodiana, which were collected from a floodplain lake of the upstream section of the Irrawaddy River basin, Kachin State, Myanmar in November 2016 during the join fieldworks with specialists of Flora & Fauna International and Department of Fisheries of Myanmar (Table 1). In addition, six new samples of S. woodiana complex from Vietnam and Russia were also sequenced. Sequences of Margaritifera dahurica and M. laosensis were used as out-groups (GenBank acc. nos. KJ161530 and KR006699, respectively).
List of the Mitochondrial COI Sequences ofSinanodonta spp. Examined in the Present Study.
Laboratory Protocols and Phylogenetic Analysis
A total genomic DNA was extracted from the alcohol-preserved mussel foot tissue using the NucleoSpin® Tissue Kit (Machereye Nagel GmbH & Co. KG, Germany), following the manufacturer’s protocol. Primers used for amplification of the COI partial sequences were LCO1490 and HCO 2198 (Folmer, Black, Hoeh, Lutz, & Vrijenhoek, 1994). The polymerase chain reaction (PCR) mix contained approximately 200 ng of total cellular DNA, 10 pmol of each primer, 200 mmol of each dNTP, 2.5 ml of PCR buffer (with 10 × 2 mmol MgCl2), 0.8 units of Taq DNA polymerase (SibEnzyme Ltd., Russia), and H2O, which was added up to a final volume of 25 ml. Thermocycling included one cycle at 95℃ (4 min), followed by 34 cycles at 95℃ (45 s), 50℃ (40 s), and 72℃ (50 s) with a final extension at 72℃ (5 min). Forward and reverse sequence reactions were performed directly on purified PCR products using the ABIPRISM® BigDye™ Terminator v. 3.1 reagents kit and run on an ABI PRISM® 3730 DNA analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The resulting sequences were checked using a sequence alignment editor (BioEdit v. 7.2.5; Hall, 1999).
The alignment of the COI sequences was performed using the ClustalW algorithm (Thompson, Higgins, & Gibson, 1994). For the phylogenetic analyses, each COI sequence of aligned data set was trimmed, leaving a 659-bp fragment. Then, identical COI sequences were removed from the data set using an online FASTA sequence toolbox (FaBox1.41; Villesen, 2007), leaving 35 haplotype sequences (including the two out-group taxa). For phylogenetic analyses, we used the COI data set with unique haplotypes. The best evolutional models for each partition calculated on the base of corrected Akaike Information Criterion through MEGA6 (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013) were as follows: (a) first codon of the COI: HKY; (b) second codon of the COI: TN93 + G (G = 0.21); (c) third codon of the COI: HKY. Phylogenetic relationships were reconstructed based on Bayesian inference using MrBayes v. 3.2.6 (Ronquist et al., 2012) at the San Diego Supercomputer Center through the CIPRES Science Gateway (Miller, Pfeiffer, & Schwartz, 2010). Four Markov chains, one cold and three heated (temperature = 0.1), were run simultaneously for 25,000,000 generations. The resulting phylogeny was constructed using a tree figure drawing tool (Archaeopteryx v. 0.9901 beta; Han & Zmasek, 2009).
A well-established population of S. aff. woodiana was discovered in a floodplain lake of the upstream section of the Irrawaddy River basin, Kachin State, Myanmar (Figures 1Figure 2. to 3). The mean shell length is 110.3 mm (90.2–128.5 mm, N = 20).
The three sequenced specimens of S. aff. woodiana belong to a single COI haplotype, which is identical to that recorded from invasive populations in Europe and from native populations in China (Figure 4). This haplotype belongs to the temperate invasive lineage of Bolotov et al. (2016), which also comprises three additional haplotypes from China. The mean COI p-distance within the temperate invasive lineage varies from 0.15% to 0.66%.
Populations from Malaysia, Indonesia, and Philippines belong to the tropical invasive lineage of Bolotov et al. (2016; see Figure 4). The sequenced specimens from Singapore are also representatives of this lineage, although they were obtained from ornamental trade (Ng et al., 2016). In contrast, the populations from Vietnam belong to a separate lineage that appears to be a native species, which inhabits the Red River basin (see Figure 4).
Among countries of Southeast Asia, representatives of S. woodiana species complex were recorded from Malaysia, Indonesia, Singapore, Philippines, Cambodia, and Vietnam but were unknown from Myanmar, Thailand, and Laos (Bolotov et al., 2016; Ng et al., 2016; Zieritz et al., 2016, 2017).
Our sequenced specimens of S. woodiana from Irrawaddy basin surprisingly reveal the same COI haplotype that was recorded from invasive European populations and that belongs to the temperate lineage of Bolotov et al. (2016).
The taxonomy of S. woodiana species complex is unclear because it includes several divergent mtDNA lineages, each of which may represent a separate cryptic species (Bolotov et al., 2016; Froufe et al., 2017). Moreover, there is a plethora of nominal taxa that were synonymized with S. woodiana. With respect to an integrative approach (Konopleva, Bolotov, Vikhrev, Gofarov, & Kondakov, 2017), a taxonomic revision of such a species complex should be based on molecular sequence data obtained from the topotypes of each nominal taxon because old museum lots with dry shells are not appropriate for the extraction of high-quality DNA. The holotype of S. woodiana was collected from the Pearl River near Guangzhou (USNM: voucher no. 86380, the type locality: “Canton, China”), but any Sinanodonta sequences from this region, which may serve as reference sequences for the species, are not available.
Finally, the genus Sinanodonta should be a focus of the extensive taxonomic revision, along with efforts to estimate the distribution of invasive lineages and to detect the environmental threats and risks from alien mussel impacts.
Implications for Conservation
The actual distribution of S. woodiana in Myanmar is still unknown, although it seems to be rather local, because it was not recorded anywhere during our extensive fieldworks across the country (Bolotov et al., 2017a, 2017b). However, its possible spread through the country may affect native benthos communities, which include many unique endemic taxa of the Unionidae (Bolotov et al., 2017b). Our survey in the Bhamo area has shown that small lakes, which harbor the majority of mussel species in this region, may represent a suitable habitat for S. woodiana. The Chinese pond mussel could successfully inhabit these waterbodies and could compete with native mussels for resources and host fishes (Bolotov et al., 2016; Zieritz et al., 2016). Negative effects of the invasive S. woodiana on freshwater ecosystem functioning have been reported from Europe and Malaysia (Donrovich et al., 2017; Sousa et al., 2014; Zieritz et al., 2016). In Malaysia, this alien species replaced the native unionid assemblages in many river systems (Zieritz et al., 2016). However, we assume that further expansion of the temperate lineage across Southeast Asia may be limited due to the specific environment of tropical floodplain freshwater systems. These environmental conditions appear too warm for this cryptic taxon, which most likely originated from temperate China (see Figure 4). In contrast, a possible invasion of the tropical lineage into large freshwater basins of Myanmar (e.g., the Irrawaddy, Salween, and Sittaung drainage basins) seems to be much more dangerous because this lineage is adapted well to the environment of tropical monsoon freshwater systems, and it may therefore spread throughout the country.
Appearance of the Chinese pond mussel in Myanmar requires regular monitoring and other environmental measures from the government and conservation organizations. Dramatic declining of native species in Malaysia as a result of S. woodiana invasion is an example of lacking of special measures for the invasion management (Zieritz et al., 2016). We assume that current local range of S. woodiana in Myanmar provides benefits for invasion control and eradication efforts.
We are grateful to Dr. Tony Whitten, Mr. Frank Momberg, Mr. Zau Lunn, and Mr. Nyein Chan (Fauna & Flora International, Myanmar) for their great help during this study. We would like to express our sincerest gratitude to the Department of Fisheries of Ministry of Livestock, Fisheries and Rural Development (Myanmar) and personally to Mr. Myint Than Soe for the permission of the field work and sampling in Myanmar (sampling permission no. 15/6000/MOAI-3103/2016 and export permission no. MOAI/2016-5856).
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was partly funded by grants from the President of the Russia Grant Council (no. MD-7660.2016.5), Federal Agency for Scientific Organizations (project nos. 0410-2014-0028 and 0409-2016-0022), Russian Foundation for Basic Research (no. 16-34-00638), and Northern Arctic Federal University.