Research Article |
Corresponding author: Christine Ewers-Saucedo ( ewers-saucedo@zoolmuseum.uni-kiel.de ) Academic editor: Adam Petrusek
© 2019 Sarah Hayer, Dirk Brandis, Günther B. Hartl, Christine Ewers-Saucedo.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hayer S, Brandis D, Hartl GB, Ewers-Saucedo C (2019) First indication of Japanese mitten crabs in Europe and cryptic genetic diversity of invasive Chinese mitten crabs. NeoBiota 50: 1-29. https://doi.org/10.3897/neobiota.50.34881
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The Chinese mitten crab (Eriocheir sinensis) is a prominent aquatic invader with substantial negative economic and environmental impacts. The aim of the present study was to re-evaluate the genetic diversity of mitten crabs throughout their native and invaded ranges based on publicly available sequence data, and assess if multiple introductions or rapid adaptation could be responsible for biologically divergent mitten crabs in Northern Europe. We assembled available genetic data of a fragment of the mitochondrial cytochrome c oxidase subunit one gene (COI) for all species of the genus Eriocheir. We applied phylogenetic and population genetic analyses to compare native and invasive populations, and to identify possible source populations. The phylogenetic reconstruction revealed that five COI sequences from Europe, morphologically identified as Chinese mitten crab, actually belong to the Japanese mitten crab (Eriocheir japonica), representing the first indication of its presence in European waters. All other COI sequences from Europe could unambiguously be assigned to the Chinese mitten crab. In some Northern German populations of Chinese mitten crabs, genetic diversity was surprisingly high, due to seven unique haplotypes encoding several amino acid substitutions. This diversity may reflect a cryptic introduction from an unsampled native location, or rapid adaptation in the invaded range. Based on the genetic diversity shared between native and introduced range, Feiyunjiang, a tributary of the Yangtze River, emerges as a plausible source population for the original introduction of Chinese mitten crabs to Europe. This study highlights the complex and dynamic invasion processes of mitten crabs in Europe. We urge to further monitor mitten crab invasions using genetic tools.
Tspecies invasion, amino acid substitution, barcoding, COI, source population, rapid adaptation
Species invasions have altered the global ecological landscape dramatically in the past centuries. Their impacts are exemplified by the Chinese mitten crab (Eriocheir sinensis H. Milne Edwards, 1853), one of the taxa included in the list of the “world’s 100 worst invasive alien species” (
Identifying the source or sources of such widespread invasions is an important task for risk assessment and species management. Assigning the geographic sources of invasive populations requires geographic differentiation within the native range. Such differentiation allowed, for example, to pinpoint the source populations of introduced olive populations in Hawaii and Australia as South Africa and western or central Mediterranean, respectively (
Since the initial assessment by
Native distribution of mitten crabs of the genus Eriocheir. Map redrawn from
Invasive populations can become melting pots of novel genetic combinations with unforeseen adaptive potential (
This could be the case for Chinese mitten crabs.
Rapid adaptation is emerging as a common feature of species invasions (
The goal of this study was to re-evaluate the genetic diversity of mitten crabs of the genus Eriocheir throughout its native and invaded ranges, and to assess if multiple introductions or rapid adaptation might have caused the recent appearance of biologically distinct mitten crabs in Northern Europe. Given a large body of previous work, we utilized publicly available mitochondrial sequence data. We employed different approaches to assign source populations to the invasive populations in Europe and the United States. First, we reconstructed phylogenetic relationships among Eriocheir sequences, grouping thereby invasive individuals into the evolutionary lineages known from the native range. In the next step, we assessed the native distribution of haplotypes also present in the introduced range. Then, we calculated genetic distances for all population pairs, which we use on the one hand to evaluate population genetic structure in the native range, and on the other hand to identify which native populations are most similar to introduced populations. We assume thereby that allele frequencies have not shifted significantly since the invasion, and that enough individuals invaded the new range to mirror native allele frequencies. Bayesian assignment tests have been proposed as a suitable alternative to assign invasive individuals to source populations (
We downloaded all available sequences for the genus Eriocheir and for two outgroup species, Neoeriocheir leptognathus and Platyeriocheir formosa (acc. nos. AF316537, AF317326;
For the phylogenetic reconstruction, we included COI sequences of all Eriocheir species and two outgroups. Most GenBank data consisted of haplotypes, not the actual sequences for each sampled individual. For the population genetic analyses, we reconstructed original haplotype frequencies by replicating haplotype sequences according to the data reported in the publications, attaching locality information to these sequences, and excluding sequences without sampling site information.
The first step was to assert the species affinities of all sequences within a phylogenetic framework. For this, we used all sequences available in GenBank and BOLD. We built a maximum likelihood tree with the PHYML (
All population genetic analyses were conducted in R version 3.3.3 (
For Chinese mitten crabs, the species with the most complex invasion history, we conducted further population genetic analyses to understand invasion patterns and get additional support for probable source populations. We compared haplotype and nucleotide diversity for all sampling sites, which we also refer to as populations. We wrote our own function to calculate haplotype diversity of each population based on the formula of
We calculated genetic differentiation between all population pairs as Φst and Jost’s D with the functions ‘pairwiseTest’ of the package ‘strataG’ (Archer et al. 2016), and the function ‘pairwise_D’ of the ‘mmod’ package (
We tested whether the native populations were sufficiently diverse to confidently assign individuals from the invaded range using the R package ‘assignPOP’ (
We extracted the DNA sequence alignment for the haplotypes, which was generated during the construction of the parsimony network (function ‘haplotype’ of package ‘haplotypes’ and function ‘write.dna’ of package ‘APE’) (
On September 25, 2018, we downloaded a total of 1020 sequences for the genus Eriocheir from GenBank, including 11 complete mitochondrial genome sequences. From these, we extracted 106 COI sequences after aligning all sequences to the complete mitochondrial genome sequence of a Chinese mitten crab from China (
The final alignment for the phylogenetic reconstruction contained 141 COI sequences (553 bp long) deposited in GenBank and BOLD under the names of the following species: 2 sequences of E. ogaswaraensis (
For the population genetic analyses, we removed the following sequences without sampling site information: AF105247, FJ455507, NC_011597, and FJ455505. The final COI dataset for population genetic analyses contained 455 sequences of Chinese mitten crabs from 45 populations belonging to 20 haplotypes and 38 COI sequences of Japanese mitten crabs from 8 populations belonging to 14 haplotypes. We reconstructed the population haplotype frequencies only for these two species because we wanted to infer the invasion sources of European and US invasions. The population-specific sequence information for Japanese and Chinese mitten crabs are summarized in Suppl. material
Our phylogenetic reconstruction recovered five main lineages of the genus Eriocheir, in agreement with previous phylogenetic studies (
The occurrence of Japanese mitten crabs outside of their native range has not been reported previously. The phylogenetic reconstruction recovered that five European individuals identified as Chinese mitten crab actually grouped with Japanese mitten crabs (Fig.
Maximum likelihood tree of mitten crab COI haplotypes. Haplotypes found in the invaded range are color-coded based on the country of collection. Numbers on branches represent bootstrap support. Branches without numbers have less than 50% bootstrap support. We omitted the outgroups from the figure to save space.
We identified 14 haplotypes for Japanese mitten crabs, labelled H1 to H14 (Fig.
We identified a total of 20 haplotypes for Chinese mitten crabs (Fig.
Parsimony networks for Chinese and Japanese mitten crabs. Each circle represents a haplotype, and the size of the circle is proportional to the abundance of this haplotype A Chinese mitten crab: The smallest circle (e.g. H8) contains a single sequence, while the largest circle (H1) contains 161 sequences B Japanese mitten crab: The smallest circle (e.g. H4) contains a single sequence, while the largest circle (H1) contains 18 sequences. The colors represent sampling sites: blue colors are native sites, yellow-orange-red colors are European sites and green colors are US sites.
In the introduced range, H2 was found in two individuals only, one sampled in the Weser river near Oldenburg and the other in the Elbe river in Brandenburg, suggestive of its overall low frequency in the introduced range (Fig.
Several Northern German populations are genetically distinct: Aukrug, Eckernfoerde, Eider, Finkenwerder, Flemhude, Schlei, and Soholmer Au (marked with an asterisk in Fig.
Geographic distribution of COI haplotype frequencies of Chinese mitten crabs. The distribution is shown in the native range (A), the United States (B), Northern Germany (C) and Europe in general (D). Haplotypes only found in the native range are colored in blue tones, haplotypes only found in the introduced range are colored in green tones, and haplotypes found in both the native and introduced range are colored in yellow and orange tones. The smallest pie chart in each graphic represents a single individual. All populations with five or more sampled individuals are named. Additionally, in a we also point out the populations of Dalian City, Wuhu and Yangtze, as they contain the otherwise rare but invasive haplotype H2. Scale bars: 250 km (A, B, D). 25 km (c).
Overall haplotype diversity for Chinese mitten crabs was 0.832, and ranged from 0 to 0.805 per population (Table
Results of population genetic analyses of Chinese mitten crab (Eriocheir sinensis) populations from sampling sites with more than five sampled individuals.
Sampling site | n | h | Haplotype diversity | Nucleotide diversity | Tajima’s D | D p-value |
---|---|---|---|---|---|---|
Native range | ||||||
Feiyunjiang, China | 10 | 3 | 0.600 | 0.002 | 0.473 | 0.636 |
Hangzhou, China | 6 | 3 | 0.733 | 0.003 | −0.057 | 0.954 |
Liaohe, China | 16 | 6 | 0.783 | 0.003 | 0.095 | 0.924 |
Nantong, Yangtze, China | 10 | 4 | 0.778 | 0.004 | 1.032 | 0.302 |
Oujiang, China | 11 | 1 | 0.000 | 0.000 | NA | NA |
Tongan, China | 8 | 1 | 0.000 | 0.000 | NA | NA |
Zhenjiang, Yangtze, China | 8 | 2 | 0.536 | 0.002 | 1.449 | 0.147 |
Vladivostok, Russia | 10 | 2 | 0.467 | 0.003 | 1.229 | 0.219 |
Geumgang, China | 15 | 2 | 0.133 | 0.000 | −1.159 | 0.246 |
Invaded range | ||||||
Thames, England | 15 | 4 | 0.714 | 0.003 | 0.666 | 0.505 |
Aukrug, Germany | 16 | 2 | 0.233 | 0.002 | −0.744 | 0.457 |
Eckernfoerde, Germany | 18 | 2 | 0.425 | 0.001 | 0.870 | 0.385 |
Eider, Germany | 45 | 5 | 0.805 | 0.005 | 1.925 | 0.054 |
Finkenwerder, Elbe | 40 | 4 | 0.729 | 0.003 | 0.758 | 0.448 |
Flemhude, Germany | 53 | 6 | 0.766 | 0.004 | 1.171 | 0.241 |
Hemmelsdorf, Germany | 10 | 1 | 0.000 | 0.000 | NA | NA |
Laascher See, Germany | 15 | 3 | 0.676 | 0.002 | 1.386 | 0.166 |
Oldenburg, Weser, Germany | 14 | 4 | 0.648 | 0.002 | 0.683 | 0.495 |
Osterholz, Elbe, Germany | 15 | 3 | 0.676 | 0.002 | 1.386 | 0.166 |
Schlei, Germany | 11 | 3 | 0.655 | 0.004 | 0.821 | 0.412 |
Soholmer Au, Germany | 36 | 3 | 0.679 | 0.005 | 2.315 | 0.021 |
Tagus, Portugal | 16 | 3 | 0.342 | 0.001 | −0.708 | 0.479 |
Sacramento, USA | 7 | 1 | 0.000 | 0.000 | NA | NA |
San Francisco, USA | 18 | 1 | 0.000 | 0.000 | NA | NA |
A total of 9 haplotypes were private. They were distributed among four native sites (Liaohe: H6, H7, H8; Nantong: H9; Vladivostok: H18, H19; Geumgang: H20) and two introduced sites (Thames: H11; Eider: H15). Estimates of population differentiation among native populations with five or more sampled individuals revealed significant population structure across the native range (Suppl. material
We used these pairwise genetic distances to identify which introduced populations were genetically similar to native populations, representing potential sources of the invasion. In general, populations dominated by the same haplotype cluster together. The two monotypic Chinese populations, Oujiang and Tongan, cluster together with the German populations from Hemmelsdorf, Tagus and Eckernfoerde (Fig.
Visualization of genetic population similarity based on pairwise genetic differences calculated as Jost’s D values. A multi-dimensional scaling plot B hierarchical cluster analysis dendrogram. Asterisks denote Northern German populations that are dominated by haplotypes not found in the native range.
The Monte Carlo cross validation procedure revealed little power to discriminate between source populations with assignment tests. The assignment accuracy averaged across replicates was 0.032. Thus, we did not attempt to assign invasive individuals to any particular native population with this method.
Amino acid substitutions took place in eight COI haplotypes: H5, H9, and H12 to H17 (Suppl. material
To our knowledge, we provide the first report of Japanese mitten crabs (Eriocheir japonica) outside their native range. Our phylogenetic reconstruction placed five sequences identified as Chinese mitten crabs clearly within the Japanese mitten crab lineage. The sequences were collected in Holland, Germany and Poland between 2009 and 2015. The German individual was collected inland in the Rhine river, and may not have necessarily migrated successfully to the North Sea for reproduction. The Dutch and Polish individuals were collected closer to the North and Baltic Sea, suggestive of an established, reproducing population of Japanese mitten crabs in Europe for the past ten years or more.
At first, it seems surprising that this invasion of Japanese mitten crabs has remained cryptic for at least a decade, but the morphological similarity between Chinese and Japanese mitten crabs did not make it obvious (Fig.
Mitten crabs collected in Holland in 2011. A Individual carrying mitochondrial DNA (mtDNA) of the Japanese mitten crab, morphologically identified as Chinese mitten crab, (BOLD accession: CBCC040-11, museum catalogue no: Universita degli Studi di Milano, Ispezione degli Alimenti di Origine Animale MALAC:00040) B Chinese mitten crab (BOLD accession: CBCC037-11, museum catalogue no: Universita degli Studi di Milano, Ispezione degli Alimenti di Origine Animale MALAC:00037). Photographs by Cristian Bernardi published under CC BY-NC 3.0 license.
Cryptic morphology is a general problem in biological invasions that can only be resolved with molecular data.
Much work has been conducted on the phylogeography of mitten crabs in their native range (
Most of the native populations had positive Tajima’s D values, albeit not significantly different from zero, which is generally interpreted as populations being in mutation-drift equilibrium. It suggests that populations did not expand, shrink, or undergo recent selective sweeps at the mitochondrial genome. This pattern of genetic stability is anticipated for native populations. That we find the same pattern in most introduced populations is unexpected. We would expect to find negative Tajima’s D values, indicative of recent bottlenecks. It seems unlikely that the introduced populations are already at equilibrium. Instead, an invasion of sufficient number of individuals that brought over a substantial amount of the native diversity could explain the observed pattern, either in a single or in multiple introduction events. In concordance with this idea, genetic diversity is not significantly lower in invasive populations, as would be expected when few individuals invade a new range.
The most distinct feature of the introduced range is the presence of seven haplotypes that have not been sampled in the native range. These haplotypes appear restricted to Northern Germany. Their distribution dominates the population structure in Europe, which divides populations with and without those unique alleles. We recovered more population structure than identified by
The US populations of Chinese mitten crabs have been speculated to be secondarily introduced from Europe (
The analyses of genetic distances between populations echo on the one hand some of the results obtained by the comparison of haplotype identity between native and invaded ranges, and highlight, on the other hand, some of the difficulties associated with population genetic analyses of non-equilibrium scenarios pervasive during invasions. We found two clusters of mixed native and introduced populations: the first cluster contained populations from across Europe and Feiyunjiang. In line with the results of haplotype identity, Feiyunjiang is therefore a plausible source population. The second cluster contains populations monotypic for the most widespread haplotype. In this case, the native populations of Tongan and Oujiang have the same genetic makeup as the introduced populations of Hemmelsdorf, Eckernfoerde and Tagus, but this similarity may well be due to small populations and strong drift in the introduced populations, which could have eradicated much of the genetic diversity. Thus the second cluster of native and introduced populations cannot be interpreted as a separate introduction.
The restricted distribution of the haplotypes H12 to H17 in northern Germany could either reflect a snapshot taken during an ongoing expansion or ecological restrictions. The most recent samples included in our analyses are those Northern German samples with unique haplotypes collected between 2008 and 2010 (
The origin of the haplotypes only found in Northern Germany remains mysterious. If we interpret the absence of those haplotypes in the Osterholz samples, and the presence of two of these haplotypes in the Finkenwerder samples ten years later as the recent and simultaneous addition of these haplotypes to Europe, the most plausible scenario is a cryptic invasion from an unsampled native site. The source of such a cryptic invasion might be located in the northern range of Chinese mitten crabs. Overall, the number of analyzed native populations was rather small given the large range of Chinese mitten crabs (Fig.
Under a scenario of multiple invasions, the amino acid substitutions we found in all of the uniquely Northern German haplotypes evolved in the native range, and were introduced during the cryptic invasion. Whether these haplotypes confer indeed a selective advantage cannot be answered with certainty. They may carry, in fact, neutral or slightly deleterious mutations but have been swept to high frequencies during a recent strong selection event at a linked region of the genome (
Alternative hypotheses to a novel introduction can explain the origin of these uniquely Northern German haplotypes. In our opinion, the second most likely explanation is that the unique haplotypes evolved in the introduced range. Given that all of these haplotypes had one to three AAS, these haplotypes might have evolved rapidly in the introduced range in response to novel ecological conditions. Moreover, all of these uniquely Northern German haplotypes are closest related to a haplotype that was already present in Northern Europe (Fig.
Lastly, we cannot ignore the fact that all sequences with uniquely Northern German haplotypes were collected by
At this point, we cannot determine if the high and unique haplotype diversity of Northern Germany is due to novel, potentially adaptive mutations that occurred after introduction, or due to multiple invasions. To clarify the origin of the unique haplotypes, we propose three approaches. Firstly, a more extensive sampling of the native range should identify if these haplotypes are present in the native range. Such sampling has already taken place (
This study uncovered complex population genetic pattern of invasive mitten crabs. Some of our findings are unambiguous, such as the presence of the mitochondrial genome of a second mitten crab species, the Japanese mitten crab, in Europe, suggesting either a cryptic invasion of this species or previous hybridization between Chinese and Japanese mitten crabs. This new European addition was only revealed by our data synthesis, which included barcoding data collected from various entities of a few individuals. The genetic diversity within European populations of Chinese mitten crabs remains puzzling, including the presence of several amino acid substitutions in haplotypes found only in Northern Germany. Taken together with the contemporaneous occurrence of a novel physiology and behavior in the same populations, it is possible that carriers of this haplotype have an adaptive advantage. Given the negative impacts of mitten crabs as an invasive species, we can only urge to monitor these invasive populations closely, using genetic tools such as the commonly used barcoding locus COI (
We would like to thank Dr. John Wares (UGA) for his improvements to the manuscripts, and his ongoing support for CES’ work. We would also like to thank the reviewers Charles Fransen and Milan Koch, as well as the Neobiota subject editor Adam Petrusek. This study was supported by a grant from the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, project number 01UQ1711).
Tables S1–S3
Data type: species data
Explanation note: Table S1. Eriocheir japonica. Table S2. Eriocheir sinensis. Table S3. Pairwise population.
Figures S1–S6
Data type: multimedia
Explanation note: Figure S1. Maximum likelihood tree of mitten crab COI sequences from GenBank and BOLD. Figure S2. Geographic distribution of COI haplotype H2 of the Chinese mitten crab (Eriocheir sinensis). Figure S3. Geographic distribution of COI haplotype H3 of the Chinese mitten crab (Eriocheir sinensis). Figure S4. Geographic distribution of COI haplotype H4 of the Chinese mitten crab (Eriocheir sinensis). Figure S5. Multidimensional scaling plot of the first and third axes based on Jost's D distances between sampling locations. Figure S6. Codons of the COI sequence of Chinese mitten crabs that translate to amino acid substitutions (AAS).