Cardamine occulta, the correct species name for invasive Asian plants previously classified as C. flexuosa, and its occurrence in Europe

Abstract The nomenclature of Eastern Asian populations traditionally assigned to Cardamine flexuosa has remained unresolved since 2006, when they were found to be distinct from the European species Cardamine flexuosa. Apart from the informal designation “Asian Cardamine flexuosa”, this taxon has also been reported under the names Cardamine flexuosa subsp. debilis or Cardamine hamiltonii. Here we determine its correct species name to be Cardamine occulta and present a nomenclatural survey of all relevant species names. A lectotype and epitype for Cardamine occulta and a neotype for the illegitimate name Cardamine debilis (replaced by Cardamine flexuosa subsp. debilis and Cardamine hamiltonii) are designated here. Cardamine occulta is a polyploid weed that most likely originated in Eastern Asia, but it has also been introduced to other continents, including Europe. Here data is presented on the first records of this invasive species in European countries. The first known record for Europe was made in Spain in 1993, and since then its occurrence has been reported from a number of European countries and regions as growing in irrigated anthropogenic habitats, such as paddy fields or flower beds, and exceptionally also in natural communities such as lake shores.


Introduction
Cardamine fl exuosa (Cruciferae) was described by Withering (1796) from the locality "Rookery at Edgbaston" in England. Recently, this name was lectotypifi ed by Post et al. (2009) by the illustration (Fascicle. 4,Table no. 48,alternatively numbered no. 277) in Curtis' Flora Londinensis or, plates and descriptions of such plants as grow wild in the environs of London (1781). Schulz (1903), in his monograph of the genus Cardamine, treated C. fl exuosa in a wide sense with a number of subspecies, varieties and formas. Out of the infraspecifi c taxa recognized by Schulz (1903), C. scutata Th unb., C. fallax (O.E. Schulz) Nakai and C. pennsylvanica Willd. are now generally recognized as separate species. Th e remaining part of C. fl exuosa had until recently been treated as a single species distributed worldwide without the recognition of any infraspecifi c taxa (Jalas and Suominen 1994, Zhou et al. 2001, Al-Shehbaz et al. 2006. It was not until the phylogenetic paper by Lihová et al. (2006) that it was realized that European and Eastern Asian populations traditionally treated as C. fl exuosa belong to two diff erent taxa. Both DNA sequence and chromosome number data demonstrated that they represent two distinct evolutionary lineages. While the native European species C. fl exuosa is tetraploid (2n = 32, Marhold 1994, Kučera et al. 2005, Eastern Asian plants, informally treated by Lihová et al. as "Asian C. fl exuosa", are octoploid (2n = 64, Lihová et al. 2006, T. Mandáková, Brno, unpublished data, Marhold et al., unpublished data, contrary to the assumed hexaploid level based on fl ow-cytometric evidence by Bleeker et al. 2008). Multiple hypotheses about the parentage of tetraploid European C. fl exuosa have been put forward, invoking both auto-and allopolyploidy (reviewed by Lihová et al. 2006 andMandáková et al. 2014). Only recently, the cytogenetic approach (combining genomic in situ hybridization and comparative chromosome painting, CCP/GISH) provided unequivocal evidence that this taxon is an allopolyploid originating from the diploids C. amara L. and C. hirsuta L. (Mandáková et al. 2014). In turn, CCP/GISH (Mandáková et al., in prep.) revealed allopolyploidy also in Eastern Asian C. fl exuosa (as inferred earlier from molecular data, Lihová et al. 2006), but with a diff erent parentage. Th ree distinct diploid genomes were identifi ed within this octoploid, corresponding to C. amara, C. parvifl ora L. (or perhaps their unknown close relatives) and another, as yet unidentifi ed taxon.
Morphological characters of Eastern Asian populations treated as C. fl exuosa and their diff erences from European populations are presented by a number of authors (e.g., Rosenbauer 2011, Hepenstrick and Hoff er-Massard 2014, Dirkse et al. 2015. Most of their descriptions, however, do not encompass the whole variation of the two taxa, and none consider diff erences from other Asian relatives, such as C. scutata, so a thorough morphometric study of C. fl exuosa and related Eastern Asian taxa is required (Marhold et al. in prep.). Th ese two taxa also show considerable diff erences in their ecological requirements. European C. fl exuosa occurs mostly in forest plant communities along wet forest roads or in various open habitats and is only seldom found as a weed in fl ower beds (often introduced with mulch of bark chips) or in greenhouses . Eastern Asian C. fl exuosa, by contrast, is primarily a weed of rice paddy fi elds, and perhaps only secondarily occurs in other open habitats (Kudoh et al. 1993, Yatsu et al. 2003. It was hypothesized by Lihová et al. (2006) that the origin and spread of this latter taxon are associated with the establishment of suitable manmade habitats (e.g. paddy fi elds). Based on morphology and molecular data, Lihová et al. (2006) reported Eastern Asian C. fl exuosa from Japan, China, Taiwan, Th ailand, Vietnam, Australia, Canada, USA and Mexico.
As a consequence, based on their genetic divergence, diff erent ploidy, allopolyploid origins, morphology, ecological requirements and distribution patterns, we are of the opinion that European and Eastern Asian populations previously treated as C. fl exuosa should be classifi ed as two diff erent taxa at the species level. Th e concept of two taxa is also adopted in the Flora of North America (Al-Shehbaz et al. 2010) and is followed by other authors reporting plants corresponding to Eastern Asian C. fl exuosa from diff erent parts of the world, particularly Europe. Several names have been used for this taxon, namely C. fl exuosa subsp. debilis O.E. Schulz (e.g., Rankin Rodríguez and Greuter 2009, Lazzeri et al. 2013, Ardenghi and Mossini 2014, Hohla 2014a, C. hamiltonii G. Don (e.g., Bomble 2014, Dirkse et al. 2015, Hohla 2015 [both replacement names based on illegitimate C. debilis D. Don (non C. debilis Banks ex DC.)] and C. occulta Hornem. (Klinkenberg 2015).
None of the above-mentioned names were properly typifi ed or used unequivocally, which necessitated a thorough search for the correct species-level name for "Asian Cardamine fl exuosa". Here we present a nomenclatural survey of all relevant names and highlight the increasing number of records of "Asian Cardamine fl exuosa" across Europe.

Materials and methods
For the purpose of typifying names, herbarium specimens, especially types and authentic collections, were searched for in relevant herbaria (B, BM, C, E, KW, LINN, P, TI and UPS), and protologues were studied in relevant publications. Bibliographical citations in databases, such as IPNI (Th e International Plant Names Index; www.ipni. org), Tropicos (www.tropicos.org) and Th e Plant List (www.theplantlist.org), were also checked, and for species, links to IPNI LSID metadata are provided. In cases when specimen images were available online, stable identifi ers for specimens (Hyam et al. 2012herbaria B, SAV), other permanent links (herbarium P) or links via JSTOR Global Plants (https://plants.jstor. org/; herbarium KW) are provided. In designating types of names of taxa, we strictly followed the International Code of Nomenclature for algae, fungi, and plants (Mc-Neill et al. 2012). We also surveyed all relevant literature sources and gathered the fi rst records of "Asian C. fl exuosa" in European countries and their larger administrative divisions.

Results and discussion
Nomenclature Th e type status of species names corresponding to "Asian C. fl exuosa" in the sense of Lihová et al. (2006) has been determined, and justifi cations for their typifi cations are presented. Cardamine occulta is the oldest name applicable to populations of "Asian C. fl exuosa". Th ere is a single specimen available in herbarium C originating from Hornemann's collection that undoubtedly represents the single remnant of the original material for the name C. occulta. As Hornemann (1819) referred to the specimen in the garden and not to the herbarium sheet, and as we cannot exclude that there was originally more than one specimen of this taxon in his collection, we designate the specimen as a lectotype of the name C. occulta (admitting that the specimen might well represent the holotype). Th e plant on the type herbarium sheet was apparently grown from seeds at the Copenhagen Botanical Garden ("ex h[ortus] b[botanicus] Hafn[iensis]"). Perhaps cultivation at the garden might be the reason why the specimen cannot be reliably and unequivocally identifi ed as "Asian C. fl exuosa" for the purposes of the precise application of the name C. occulta to this taxon (especially considering the occurrence of a number of closely related taxa in China; Zhou et al. 2001). Th erefore, in order to fi x the application of the name C. occulta, we designate here an epitype of this name from a cytogenetically investigated population from Eastern China with a known chromosome number (2n = 64; Mandáková et al., in prep. Th e name C. debilis D. Don is based on data in the manuscript of Francis Buchanan-Hamilton (referred to as "Hamilton MSS"; Don 1825: 201), and it is unclear whether D. Don studied any specimen collected by Buchanan-Hamilton. Although Hara and Williams (1979) mentioned the type of C. debilis [when indicating localities of C. scutata subsp. fl exuosa (With.) Hara in Nepal], in Shrestha and Press (2000), the type specimen is listed as "not found". In any case, a thorough search in the herbaria BM, E, LINN-Smith (Roy Vickery, John Edmondson, Mark Watson, personal communication) did not reveal any original material of this name. Th ere is a specimen corresponding to the description of C. debilis D. Don and to "Asian C. fl exuosa", collected in the neighbouring area of West Bengal, with a chromosome number counted by B. Lövkvist (2n = 64, unpublished data, deposited at UPS). Th is specimen is selected here as a neotype to fi x the application of the name. Th e species C. autumnalis was described with a reference to "Cardamine fl exuosa ssp. debilis Schultz (pro. parte) in Engl. Bot. Jahrb. 32. (1903) s. 479, (quoad specim. ex Yokoska)". Indeed, there is a specimen marked "Japonia: … pr. Jokohama leg. Wichura 1860" referred to by Schulz (1903: 479) as Cardamine fl exuosa subsp.

Cardamine occulta
debilis deposited in B. Th e specimen bears a revision label by Schulz with the name "Cardamine fl exuosa With. subspec. debilis Don var. occulta (Hornem.) O. E. Sch.", dated 25. 4. 1902. Although this specimen was identifi ed by Schulz as var. occulta, it should be noted that there is no specimen referred to by Schulz (1903: 480) identifi ed as Cardamine fl exuosa subsp. debilis var. occulta from Japan.
Th e usual life cycle of C. occulta in Eastern Asian rice fi elds includes fl owering in early spring before rice is planted and the fi elds are fl ooded by water. Nevertheless, there are also exceptions such as the nomenclatural type of the name C. autumnalis, which represents an autumn-fl owering plant of C. occulta. Kudoh et al. (1993: fi g. 8) reported such plants from paddy fi elds in the autumns of years in which rice was not cultivated (no water fl ooding during summer).
Th ere are two other names at the species level that are potentially applicable to "Asian C. fl exuosa", namely: Cardamine nasturtioides D. Don, Prodr. Fl. Nepal.: 201. 1825 Th e location of the original material of these two names is as yet unknown, and it remains to be ascertained whether they are synonyms of C. occulta or represent other taxa. In any case, both these names are later than C. occulta, which has priority among all species names applicable to "Asian C. fl exuosa".
Th e name C. zollingeri Turcz. was sometimes considered to be a synonym of C. fl exuosa in a wide sense (e.g., Zhou et al. 2001, Al-Shehbaz et al. 2006, Al-Shehbaz and Watson 2012) or of C. fl exuosa subsp. debilis (Schulz 1903: 479). Nevertheless, it is morphologically diff erent from both C. fl exuosa and C. occulta in the circumscriptions presented here and likely represents a separate taxon that requires further study: Cardamine zollingeri Turcz., Bull. Soc. Imp. Naturalistes Moscou 27 (2)

Occurrence of Cardamine occulta in Europe
Cardamine occulta most likely originated in Eastern Asia. It is unclear whether it naturally occurs or ever occurred in any natural plant community. Th e localities that we know from Japan and Eastern China and which are referred to on herbarium specimens represent solely man-made habitats, most often rice paddies, orchards or various other kinds of synanthropic vegetation. Th is is why we  hypothesized that the origin and spread of this polyploid species might have been connected with the occurrence of man-made habitats. As stated above, Lihová et al. (2006) reported plants corresponding to C. occulta from Japan, China, Taiwan, Th ailand, Vietnam, Australia, Canada, USA and Mexico. Other previously published data corresponding to C. occulta than those that were referred to by Lihová et al. (2006) were the report of C. debilis D. Don from North America as an introduced weed (Rollins 1993) and C. aff . fl exuosa from Australia (Th ompson 1996). Subsequently, this taxon was published also for Cuba (Rankin Rodríguez and Greuter 2009, as C. fl exuosa subsp. debilis).
When Lihová et al. (2006) suggested that European and Asian C. fl exuosa should be treated as separate taxa, no record corresponding to Asian C. fl exuosa plants was known from the European territory. Nevertheless, a number of records from Europe have been published since 2007, and we can trace the spreading of this invasive plant throughout the continent (see Table 1, Fig. 1). To the best of our knowledge, the earliest record of C. occulta from Europe dates back to 1993, when this species was collected in the Spanish province of Alicante and originally identifi ed as C. fl exuosa. Its true taxonomic identity was, however, clarifi ed much later (Crespo et al. 2013). In 2007 the fi rst author of this paper received for identifi cation a specimen collected in 2003 in a rice fi eld ditch in the province of Piedmont, Italy (Vercelli, Arborio) by Michel Desfayes (Fully, Switzerland). Th is specimen undoubtedly belongs to C. occulta and might have been introduced together with rice from Eastern Asia. From the same broad locality, the occurrence of this taxon was reported by Th omas Götz (a specimen collected in 2005, published by Dienst 2007) and more recently by Verloove and Ardenghi (2015;as C. hamiltonii).
Th e third spot in Europe where C. occulta was reported from are the shores of Lake Constance (Bodensee) in Germany. In spring 2004, an unknown Cardamine species was detected there at the Reichenau dam (observed by W. Ostendorp, M. Dienst and E. Klein;Dienst 2007). Th e identity of these plants was confi rmed by DNA sequenc- Table 1.  ing (Bleeker et al. 2008). Until 2007, 95 locations on the shores of Lake Constance had been known. Localities were found around the lake in Germany (Baden-Württemberg and Bavaria), Austria (Vorarlberg) and Switzerland (cantons Schaff hausen, Th urgau, and St. Gallen; Bleeker et al. 2008). Bleeker et al. (2008) noted that C. occulta was more frequent on fi ne-grained and nutrient-rich sediments than on nutrientpoor gravel shores. It is likely that this species may change the community structure of ephemeral vegetation on bare and organic sediments. Cardamine occulta was later reported also from continental Spain, the Canary Islands, France, parts of Germany, Switzerland and Austria other than the shores of Lake Constance, from Belgium, the Netherlands, Slovakia, and Crete (Table 1). It is nevertheless likely that the species is currently present, but still overlooked, also in other European countries. It should be noted that most records mentioned in Table  1 refer to urban vegetation. Cardamine occulta grows in fl ower beds and pots, at the edges of roads, among cobblestones or paving stones, or on pavements, often in irrigated places. In most cases, it was apparently introduced as a weed, often with mulch, from plant nurseries where it fi nds appropriate growing conditions (as reported from North America by Post et al. 2011). However, the species was also found in rice fi elds in northern Italy, where it was most likely introduced with rice from Eastern Asia.
Th ere are only a few known occurrences of C. occulta in European natural plant communities, and it seems that such reports are restricted to the surroundings of Lake Constance. Bleeker et al. (2008) hypothesized that this species might have been introduced to the lake from rice fi elds of northern Italy by migrating birds or directly from Japan by tourists.
For most of the countries and administrative divisions presented in Table 1, only one or few localities of C. occulta are known. Th ere are numerous observational records of C. occulta from the Netherlands and Belgium in the databases presented at observation.org, waarneming.nl and waarnemingen.be (referred to as C. hamiltonii), perhaps because botanists in these countries were encouraged to searched for it. Nevertheless, there are no voucher specimens documenting these data, and some of them are not even documented by photographs. According to the photographic documentation, some records are apparently based on misidentifi cations of C. hirsuta and tetraploid C. fl exuosa. A number of photographic records document juvenile plants that are hard to identify reliably. For future mapping of the distribution of C. occulta, all records should be documented by vouchers deposited in public herbaria.
It is apparent that, unlike European C. fl exuosa, C. occulta represents an invasive species that is quickly spreading from its area of origin in Eastern Asia to other continents. Th e characteristics of seed dormancy and germination of C. occulta are likely to enhance its invasiveness, especially in wet and occasionally submerged habitats. It has been reported that seeds of C. occulta can survive both in dry and submerged conditions for more than three months (Yatsu et al. 2003). Th e combination of seed dormancy in dry soil and dormancy release by submergence (Yatsu et al. 2003) is likely to enhance the transportation of C. occulta seeds with soils and the establishment of invasive populations in seasonally submerged habitats such as paddy fi eld, dams or lake shores and in regularly irrigated fl ower beds and other urban habitats. Diploid C. hirsuta is in fact another example of the invasive potential of Cardamine species. Th is species originated in Europe and is now widely distributed on all continents, particularly in drier conditions. Th e speed of its spreading can be illustrated on the example of the Japanese archipelago. While the fi rst record of this species for Japan dates to 1974 (Kudoh et al. 1992), already in 2006 it became a common roadside weed across most of Honshu Island, the main island of Japan, and was spreading also to Kyushu and Hokkaido Islands (Yatsu et al. 2003, Kudoh et al. 2007).