Invasive records of Eriocheir hepuensis Dai , 1991 ( Crustacea : Brachyura : Grapsoidea : Varunidae ) : Implications and taxonomic considerations

A non-ovigerous female mitten crab captured in Iraq on the 20th June 2005 was initially identified as Eriocheir sinensis H. Milne Edwards, 1853. More material has now been made available from Iraq and Kuwait and these specimens were compared with the extant type series of E. sinensis and E. hepuensis Dai, 1991. From this morphological study the Persian Gulf material was identified as the Hepu mitten crab, E. hepuensis. This is the first report of this mitten crab species outside its native range of southern China. However, because mitten crab taxonomy and systematics requires further clarification especially with respect to species of Eriocheir De Haan, 1835, the DNA of the Persian Gulf material was compared with a suite of GenBank COI sequences from various mitten crab taxa. The results of this extensive examination indicate that the Iraqi and Kuwaiti specimens are E. hepuensis and the DNA analysis indicates that mitten crabs can be assigned to three genera and six species; although the present study does discuss reports of hybridisation between three Eriocheir species associated with aquaculture. Also reported, and of concern, is that E. hepuensis may have been first collected about thirty years ago in Iraq and has now become well established within that country. As a consequence the Hepu mitten crab could, given its catadromy, disperse widely throughout the extensive riverine systems of the region.


Introduction
Four species of "hairy" (colloquially Southeast and East Asia) or "mitten" (vernacular Europe) crabs are currently assigned to Eriocheir De Haan, 1835 (Brachyura: Grapsoidea: Varunidae), initially, E. japonica (De Haan, 1835), and E. sinensis H.Milne Edwards, 1853, and recently, E. hepuensis Dai, 1991, and E. ogasawaraensis Komai, Yamasaki, Kobayashi, Yamamoto and Watanabe, 2006(Ng et al. 2008).Eriocheir species are indigenous to China, the Korean Peninsula and Japan (Figure 1), and are all catadromous, spending most of their lives in fresh water, but returning to higher salinity water for mating, spawning and larval development.The seaward migration is annual, occurring during the autumnal months, and after mating the adults die (semelparous).The life cycle is completed by the upstream migration of early crab stages into freshwater.
Eriocheir sinensis has become arguably the most notorious brachyuran species on the planet.It is one of only two crabs on the world's list of 100 most invasive aquatic invertebrates (Lowe et al. 2004).Incidentally, the other brachyuran species listed is the portunid Carcinus maenas (Linnaeus, 1758).The Chinese mitten crab is also cited in the handbook of alien species in Europe (DAISIE 2009: 312).This crab has successfully invaded NE Europe and has been reported from the east (Ruiz et al. 2006;Dittel and Epifanio 2009;Schmidt et al. 2009) and west coast (Cohen and Carlton 1997) of the United States and eastern Canada (Nepszy and Leach 1973;de Lafontaine 2005) with records from southern Iraq (Clark et al. 2006;Hashim 2010), and Tokyo Bay, Japan in 2004 (S.Kobayashi,  (pers.comm.; Doi et al. 2011).Furthermore, E. sinensis is one of the most commercially valuable crabs in East and Southeast Asia (e.g., see Peng 1986;Zhao 1988;Ng 1998;Lai et al. 1992), where the gonads, which develop during the annual downstream migration, are regarded as a delicacy (Figure 2).
Eriocheir japonica is also commercially exploited, but not to the same extent as E. sinensis, being consumed predominantly in Japan.The Japanese mitten crab too has been reported from outside its home range when a solitary male crab was captured in the Columbia River approximately 3km west of the Astoria-Megler Bridge, Astoria, Oregon, USA (see Jansen and Armstrong 2004).No further exotic reports have been recorded.
Recently, new mitten crab specimens from a number of localities in Iraq and Kuwait have been reported (Figure 3).Some of this material, which included several large males, has become available for detailed examination.The specimens were identified as Eriocheir hepuensis, the Hepu mitten crab.This is the first report of the crab outside its native range of southern China.However, because mitten crab taxonomy and systematics requires further clarification especially with respect to species of Eriocheir De Haan, 1835, a more extensive investigation was undertaken.This included a thorough examination of available E. sinensis and E. hepuensis material, a morphometric analysis, and the DNA barcode (sensu Costa et al. 2007; the mitochondrial COI gene) of an Iraqi mitten crab was compared against a suite of GenBank sequences for Eriocheir.
The present study reports upon the results of the morphological and genetic investigations of the mitten crabs from the Persian Gulf, and the implications for mitten crab taxonomy and systematics.Also discussed is the capacity of E. hepuensis for further dispersal throughout the extensive watersheds of the region.Literature: Only a selected synonymy for E. sinensis and E. hepuensis is provided here, but see Guo et al. (1997) for additional references.
PCR amplifications were 12.5µl reactions on a Geneamp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) of 0.5µl template DNA, 0.4µM primers, 0.1µM dNTPs, 2µM MgCl2, 2.5µl 10X PCR Buffer, 0.5 units of Taq polymerase (Bioline Pty Ltd, Alexandria, NSW, Australia) and the rest ddH 2 0. The following cycling conditions were used: 15 cycles of 30 s at 94°C, 30 s at 40°C, 60 s at 72°C; 25 cycles of 30 s at 94°C, 30 s at 55°C, 60 s at 72°C.BigDye v.3.1 Terminator mix (Applied Biosystems) was used for the sequencing reaction in both directions and the sequences produced on an Applied Biosystems 3130xl Genetic Analyser at the DNA Sequencing Facility at Griffith University.Sequences were edited using Sequencher 4.1.2(Gene Codes Corporation).
The COI sequence was compared against all GenBank sequences (17 Apr.2011) using the BLASTN search at blast.ncbi.nlm.nih.gov, and also compared against the Barcoding of Life (BOLD) online database at http://www.barcoding life.org.All GenBank Eriocheir COI sequences were downloaded and aligned with our sequence and a minimum evolution tree created using PAUP* version 4.0 b10 (Swofford 2002) with a Kimura 2-Parameter model (K2P), and bootstrapped 1000 times.A haplotype network was constructed with TCS version 1.21 (Clement et al. 2000) using sequences closely related to the Iraqi specimen (>99%) to explore withinspecies phylogeographic patterns.Molecular distances (Kimura 2-Parameter) between and within groups were calculated using Mega version 2.1 (Kumar et al. 2001).Table 1.List of characters that can be used to distinguish between Eriocheir sinensis and E. hepuensis.

Characters/Species E. sinensis E. hepuensis
Teeth on frontal margin Acutely triangular, sharp and high (Figure 5a) Broadly triangular and sometimes not as sharp, and not as high (Figure 5b) Median cleft on frontal margin Narrow v-shaped (Figure 5a) Broad v-shaped (Figure 5b) Carapace physiognomy High and convex High but not convex Anterolateral teeth of carapace Usually sharp and strong (Figure 6a) Weaker and not as sharp (Figure 6b) 4th leg merus Long and slender (Figure 7a) Short and broad (Figure 7b) 4th leg propodus Long and slender (Figure 7a) Shorter and broader (Figure 7b) Male G1 Gonopore close to distal end Gonopore about ½ length from distal end Female gonopore Semicircular in shape, concave dorsally Triangular in shape, slightly concave dorsally  (Rathbun, 1913) and Platyeriocheir formosa (Chan, Hung and Yu, 1995).However, this classification and the present identification of the Hepu mitten crab specimens from the Persian Gulf may be considered as controversial to some researchers (Tang et al. 2003(Tang et al. , 2004;;Sun et al. 2003Sun et al. , 2005) ) who consider E. hepuensis as an invalid species.
They consider the Hepu mitten crab to be a junior synonym of E. sinensis.Furthermore, Chu et al. (2003) have argued for maintaining five species (E.ogasawaraensis was described later) within Eriocheir, while Tang et al. (2003) suggested that E. japonica, E. sinensis and E. hepuensis were conspecific and should be considered as subspecies of E. japonica.In contrast, studies on morphology (Guo et al. 1997;Li and Zheng 2000a), reproductive biology (Li and Zheng 2000b), geography and habitats (Li and Zheng 2001), as well as a cladistic analysis based on 46 adult morphological characters (Ng, N.K. unpublished data; Figure 11) show that the Hepu mitten crab should be recognized as a separate taxon from E. sinensis.Interestingly, Figure 11 does not support a single taxon as suggested by Tang et al. (2003Tang et al. ( , 2004)) teeth, the shape and position of the carapace ridges (Figure 4, 5, 6), the meral and propodal proportions of the fourth ambulatory leg (Figure 7) and the position of the genital pore on the male gonopod (Figures 8, 9).

Molecular
As the validity of E. hepuensis appears to be divisive, a DNA analysis was undertaken to further clarify mitten crab systematics.For the present study, a 659 base pair COI sequence was produced (GenBank accession number JF81 0991) which corresponds to positions 5343-6001  2009), but also included sequences from Tang et al. (2003) and Chu et al. (2003) and some unpublished sequences.The final alignment of all Eriocheir sequences was 554 base pairs.The Iraqi specimen formed part of a strong clade (bootstrap 100%) in a minimum evolution tree (Figure 12a) that includes all 21 closely related sequences (average 0.3% K2P molecular distance within the clade).This clade contains specimens listed on GenBank as being E. hepuensis, E. japonica and E. sinensis.Since different authors consider these Eriocheir taxa in many different ways (as separate species, separate subspecies or all conspecific), these sequences are labelled on the tree as the "E.hepuensis" clade on the basis of their geographic location rather than as a formal taxon.Essentially, there were seven groups in the tree (Figure 12a).Neoeriocheir leptognathus and Platyeriocheir formosa were distinct from each other and all other taxa.The remaining five groups form a strong clade, but the relationship between them is uncertain.These groups consist of 1) E. japonica from mainland East Asia which corresponds to the species in the strict sense as this includes the type locality of mainland Japan; 2) E. ogasawaraensis from the Ogasawara Islands; 3) E. sinensis which is the species in the strict sense as it includes the type locality of mainland northern China; 4) "E.japonica" from Okinawa, which may represent a cryptic species (or a geographic lineage of one of the other species); and 5) a mix of "E.sinensis", "E.japonica" and "E.hepuensis", including the specimen from Iraq, here referred to as the "E.hepuensis" clade.
The mixed nature of the last group can be explained by a number of factors (which are not mutually exclusive).Firstly, the GenBank specimens may have simply been identified incorrectly.This is more likely if only juveniles were used, purchased mixed from markets and collectors, and/or the worker was inexperienced with Eriocheir taxonomy.Some of the taxonomic characters used to separate the different species may also be less reliable due to possible variation.Consequently, new characters may need to be considered.Table 1 presents morphological characters currently used to distinguish between E. sinensis from E. hepuensis.In any case, juveniles and immature specimens can be difficult to separate (see Guo et al. 1997).In addition, the two species are genetically close enough to hybridise and this would make definitive species level identification difficult.If sorted by DNA barcoding then the groupings would be determined by the species of the mother (because COI is a mitochondrial gene so only inherited maternally).A recent paper by Sui et al. (2009) covering six river systems using six microsatellite loci nevertheless support the species distinction of E. japonica, E. sinensis and E. hepuensis as recognised by Dai (1991), but does hint at possible hybridisation.
The records of E. hepuensis and E. japonica from coastal southern and central China (~21°40'N to ~32°N) and Taiwan (see cited localities in Xu et al. 2009) need to be reexamined using freshly collected wild specimens.Interestingly, Eriocheir hepuensis haplotypes were the only species identified from  coastal southern China.In coastal central China, both E. hepuensis and E. sinensis were present.Eriocheir sinensis dominates northern China, with only a single E. hepuensis mitochondrial haplotype found in the Chiagjiang River (Hu and Wang, unpublished data).Sui et al. (2009) found nuclear DNA evidence of a split between northern and southern coastal China (Figure 1).
A haplotype network was created using sequences from the E. hepuensis clade (Figure 12b).Five geographic areas were defined: Coastal China South (Hepu to Aotou), Coastal China Central (Tongan, Minjiang [see Tang et al. 2003], Feiyunjiang River, Ouijiang River), Coastal China North (Chiagjiang River, Hu and Wang unpublished) and Taiwan (Tamshui).There is little obvious geographic structuring between the areas, with haplotypes shared between many areas.The Iraqi haplotype was unique and separated from the common central haplotype by two base pairs.It is not possible to narrow down the native geographic origin of the Iraqi mitochondrial haplotype because there is little geographic structuring among the areas.

Commercial exploitation and hybridisation
Eating the developing mitten crab gonads has been part of Chinese culture for many centuries dating back to the Ming Dynasty (1368-1644), when skilled craftsmen made a set of gold utensils for eating a crab that included a mallet, scissors, a shell cracker, a round salver, scoop, spoon, a long fork and combined scraper and pricker (http://www.cultural-china.com/chinaWH/html/en/Kaleidoscope2789bye8009.html).
Mitten crabs have also been a popular subject for beautiful brush-stroke paintings depicting the consumption of this delicacy (Figure 13).Today, mitten crabs command a high price in SE Asian restaurants (e.g., ca.$40 for a single crab in the right condition, Figure 14) during the autumnal months when they are harvested during their  annual migration from freshwater rivers to brackish estuarine water and marketed.Typically while in season, Chinese mitten crabs can be purchased from street markets as in Hong Kong (Figure 15).However, wild Chinese populations have dramatically declined due to overexploitation, increased demand, river pollution and irrigation schemes that have disrupted the natural migration patterns of this species (Hymanson et al. 1999).But, local and international demands for E. sinensis have been met by an intensive aquaculture programme and this species has been farmed throughout China for the last 40 years (Sui et al. 2009) especially along the Yangtze valley (Jin et al. 2001;Wang et al. 2006).This industry is estimated to be worth ca.US$ 1.25 billion annually.
Studies on growth rates for the Hepu mitten crab, revealed maximum growth at water temperatures between 23 and 28°C (N.K. Ng, unpublished data).At this range of temperatures, E. hepuensis growth is better than that known for E. sinensis, and therefore in order to enhance the stock, farmers have cross-bred different genetic populations from various parts of China, including hybridising E. sinensis with E. hepuensis (see Zhao et al. 1988;Y. Cai, pers. comm., 1999).Although offspring have been produced in these E. sinensis and E. hepuensis crosses, there are no reports of their viability (Y. Cai, pers. comm. 1999;S. Yang, pers. comm. 2000).Furthermore, some farmers in northern China have also imported southern populations (including E. hepuensis) for breeding programmes (A.-Y.Dai, pers. comm. 1998).However, because farmers did not recognise E. hepuensis and E. sinensis as separate species but only as geographic populations, they did not keep records as to the sources of the breeding pairs.Moreover, the breeders did not check if the F1 generation were fertile.In addition, cross breeding between E. sinensis and E. japonica has been attempted in South Korea recently (S.H. Ko, pers.comm.), but again there are no reports on the outcome of this genetic experiment.One problem resulting from this practice is that according to S. H. Ko (pers. comm. 2002) there appears to be no 'pure' South Korean populations of E. sinensis left in the wild.In Japan, however, no interbreeding experiments of E. sinensis and E. japonica are known to date (T. Gao 2002;S. Kobayashi, pers. comm. 2010).
As a consequence of cross breeding, the taxonomy of mitten crabs native to East Asia has become even more problematic and controversial.This is why some taxonomists do not recognise E. hepuensis as a valid species.Even the validity of species like E. japonica and E. sinensis has been questioned because the genetic and morphological characters do not always agree.The presumed natural distributions (see Figure 1) of the four known Eriocheir species have been discussed at length by Guo et al. (1997) and Komai et al. (2006).Interestingly, E. hepuensis is currently considered locally to be an endangered species in China (Cheng et al. 2009).
The first actual record of a "mitten crab" in Iraq may date back to 1980 when S.D. Salman (pers.comm. in letter to JM Bishop dated 17.04.06)reported possible collection of mitten crabs from the Shatt Al-Arab and later from the "Marshes", south of Al Fao, Shatt Al-Arab and Shatt Al-Basrah.This suggests that there has already been a long period (over 30 years) of establishment which includes ovigerous material.A rapid expansion of range could be expected as three rivers feed the Shatt Al-Arab waterway in Iraq -the Euphrates, Tigris, and Karun (see Clark et al. 2006).The origins of the Euphrates and the Tigris are in eastern Turkey and both flow to the Persian Gulf via Syria and Iraq.The Euphrates River is approximately 2800 km in length compared with the Tigris River, ca.1800 km.Rising in the Zagros Mountains, West Iran, the Karun River flows south for about 720 km to meet the Shatt Al-Arab waterway on the Iraqi border (see Figure 3).Due to the catadromy of mitten crab species, all three watersheds would be vulnerable and these distances are within the range of those already reported for E. sinensis.

Origins and vectors
The Eriocheir hepuensis specimens reported from Kuwait (Figure 3) may just be Iraqi mitten crabs that have moved westward along the coastline or down the Shatt Al-Basrah Canal.As Kuwait has no riverine system, this species it not expected to become established in the sheikhdom.However, with regard to the origin of E. hepuensis from Khor Abdullah (Figure 3), it is possible that the species was introduced to the Gulf region by ship traffic.Either adult or subadult specimens may have been transported on ship hulls or larvae with ballast water.The latter mechanism has been identified as the main pathway for dispersal of Eriocheir species as well as for other marine invasive species (Cohen and Carlton 1997;Minchin and Gollasch 2002).
Commercial vessel traffic in the Persian Gulf is heavy and increasing.During the 365-day period from May 2006 through April 2007, 44,845 commercial vessels entered the Gulf for a daily average of 123.This represents a 40% increase over the previous year (Capt.A.M. Al-Janahi, Marine Emergency Mutual Aid Centre, Bahrain, pers. comm., 9 Oct. 2007).Many of these vessels were tankers, which are empty and discharge ballast water as they load crude oil.Although records of mitten crabs from the Gulf have only recently been published (Apel and Bishop 2006;Clark et al. 2006), their presence in the upper Gulf's Shatt Al-Arab area has been known since 1980 (S. Salman, Basrah University, pers. comm., 17 Apr. 2006), and a "very large sample" of mitten crabs was captured by trawl some miles south of Iraq's Fao Peninsula.Salman also reports capturing mitten crabs in the Iraqi marshes and the Shatt Al-Arab (pers. comm., 17 Apr. 2006).As the world's demand for crude oil increases, so will tanker traffic, and re-introductions of this species or introductions of other exotic species is likely.

Conclusion
The identification of Eriocheir hepuensis from Kuwait and Iraq based on morphological characters was confirmed by DNA analysis.Furthermore, these molecular data supports the view that mitten crabs comprise three genera and six species i.e., Eriocheir japonica, E. sinensis, E. hepuensis and E. ogasawaraensis, Neoeriocheir leptognathus and Platyeriocheir formosa.However, hybridisation between E. sinensis/ E. hepuensis and E. japonica/E.sinensis are reported from aquaculture experiments.Furthermore, evidence presented here suggests that E. hepuensis may have been present for over thirty years and has now become well established in Iraq.Considering the natural distribution of E. hepuensis and its congeners, it appears that the Hepu mitten crab inhabits subtropical and tropical regions, while E. sinensis is restricted to more temperate climates (Figure 1).Therefore E. sinensis has successfully invaded mainly temperate regions in central and northern Europe and North America and E. hepuensis might be much better adapted for the subtropical climate in the Gulf region, thus implying that an even larger area of the world may be at risk of Eriocheir invasions.

Figure 2 .
Figure 2. Advertisement in a local newspaper promoting mitten crab on the menu of the Hôtel Plaza Athénée, Bangkok for the whole of November, 2001.

Figure 8 .
Figure 8. Morphology of the first male gonopod of three mitten crab species (from Guo et al. 1997).

Figure 12 .
Figure 12.A) Minimum evolution tree of Eriocheir COI sequences with bootstrap support values (number of sequences in parentheses); B) Haplotype network of "E.hepuensis" clade showing geographic origin of relevant specimen.

Figure 13 .
Figure 13.Chinese art depicting cooked Eriocheir sinensis waiting to be eaten.By Meishuaihaozhe Zhiyou, Courtesy Jiangsu Fine Arts Publishing House.

Figure 14 .
Figure 14.Chinese mitten crabs for sale at the Turf City Seafood Restaurant, Bukit Timah Road, Singapore.Note usual price for 250grm (or above) male is $65SD = ca.$33US.Photograph taken by Peter Ng.

Figure 15 .
Figure 15.In season, Chinese mitten crabs tied-up in rice grass can be purchased from street markets in Hong Kong.Photograph taken by Phil Crabb, NHM Photo Unit.

Table 2 .
Eriocheir COI sequences downloaded from GenBank that are 99% similar to Iraqi specimen.

Table 3 .
between Eriocheir groups from Figure15a(numbers in parentheses are within group averages).
sequences), all belonging to Eriocheir.Most of these were derived fromXu et al. ( Zhao N, Du N, Bao X,Zhang L (1988)Artificial Breeding, Propagation and Culture of Chinese Mitten Crab.Anhui Science and Technology Press, Hefei, People's Republic of China, pp 134-136 Zibrowius H (1991) On going modification of the Mediterranean marine fauna and flora by the establishment of exotic species.Bulletin du Muséum d'Histoire Nataturelle de Marseille 51: 83-107 Appendix 1.Other Eriocheir COI sequences downloaded from GenBank (<99% similar to Iraqi specimen).