First records of non-native species Callitrichedeflexa (Plantaginaceae), which was previously misidentified as C.terrestris in Japan

Abstract Background The cosmopolitan genus Callitriche (Plantaginaceae) is a clade of small herbaceous plants that encompasses terrestrial and aquatic species. In Japan, six Callitriche species have been identified: four native and two naturalised species. Callitricheterrestris, a naturalised terrestrial species, was first reported in 1984 in Kanagawa Prefecture and it is thriving today. New information We report the presence of a new naturalised terrestrial species, Callitrichedeflexa, which has been previously misidentified as C.terrestris because of its similar morphology. Callitrichedeflexa can be distinguished from C.terrestris through genetic differences and distinct morphological traits, such as longer pedicels. Re-examination of herbarium specimens in the Kanagawa Prefectural Museum of Natural History confirmed that most of the specimens labelled as C.terrestris, including voucher specimens from the original report, were indeed C.terrestris, but a few were C.deflexa. We also noted that the plants referred to as “C.terrestris” in our previous developmental studies should be corrected to C.deflexa.


Introduction
The genus Callitriche L. belongs to the family Plantaginaceae and is composed of approximately 50-75 species (Lansdown 2008).The genus comprises primarily small, herbaceous species, many of which are aquatic in nature.Callitriche is often considered a challenging taxon to identify because of substantial variations in gross morphology in response to different environments, as well as an extremely simplified flower morphology (Lansdown 2008).Like many aquatic plants, some Callitriche species exhibit significant morphological variations in leaves and stems even within a single individual, as they are influenced by the aquatic habitat (Deschamp and Cooke 1985, Jones 1955, Koga et al. 2020).Therefore, flower and fruit characteristics become especially important for the identification of different Callitriche species.However, the Callitriche flower is achlamydeous and diclinous, consisting of only a carpel or stamen and lacking showy petals and sepals (Erbar and Leins 2004).These organs are often microscopic, making taxonomically relevant traits difficult to discern.Overall, these characteristics contribute to the difficulty in the accurate identification of Callitriche members.
In Japan, four native Callitriche species are currently recognised: Callitriche hermaphroditica L., Callitriche japonica Engelm.ex Hegelmaier, Callitriche palustris L. and Callitriche fuscicarpa Lansdown (Lansdown 2006b).In addition, Callitriche terrestris Muhl.ex Raf.(Katsuyama and Kawasumi 1999) and Callitriche stagnalis Scop.(Morita and Lee 1998) have been reported as naturalised species (Uemura et al. 2015).Callitriche terrestris is native to North and South America (Lansdown and Hassemer 2021) and it has been reported as a non-native species in Europe (Lansdown 2006a, Lansdown 2008, Lansdown et al. 2017b).Callitriche stagnalis is an aquatic species native to Europe and it is also a widespread alien species in various other regions worldwide, such as North and South America (Philbrick and Jansen 1991, Lansdown 2009, Hassemer and Lansdown 2018) and Australia (Bean 2007, Lansdown 2022).
A recent molecular phylogenetic study of Callitriche species, including Japanese specimens of some of the species mentioned above, revealed that C. japonica is a sister of the other major members of this genus, whereas C. hermaphroditica, C. stagnalis and C. palustris belong to distinct clades (Ito et al. 2017).The phylogenetic position of C. terrestris found in Japan has not yet been examined.On the basis of these phylogenetic relationships, several comparative developmental studies with the Callitriche species found

Phylogenetic analysis
To evaluate the phylogenetic positions of the samples, rbcL and matK sequences of Callitriche and some outgroup species were obtained from NCBI/DDBJ/EBI.The accession numbers are listed in Suppl.material 2. We used the data from Ito et al. (2017), which provided both rbcL and matK sequences from a broad range of Callitriche species.DNA alignment was conducted by MAFFT (Katoh and Standley 2013).The Maximum Likelihood tree was reconstructed using concatenated sequences of matK and rbcL by IQ-TREE 2 ( Minh et al. 2020).The confidence values of each node were calculated using both ultrafast bootstrapping and Shimodaira-Hasegawa-like approximate likelihood ratio test (Hoang et al. 2017).

Keys to identification of Callitriche species recorded from Japan
We provide identification keys for the species level.See Lansdown (2006b)

Analysis Morphological traits
On the basis of recent descriptions (Lansdown et al. 2017a, Lansdown et al. 2017b, Hassemer and Lansdown 2018, Lansdown and Hassemer 2021), morphological traits of the collected plants, particularly flowers and fruits, were examined.The plants were raised in a growth chamber and harvested before observation.The specimens were divided into two types.One type, represented by specimens KKmz1, KHsm1 and IbTcu1, displayed traits consistent with those of C. terrestris, as described in an original report from Japan (Katsuyama and Kawasumi 1999), including short pedicels (≤ 1 mm) that point downwards when the fruits mature (Fig. 1B, C).It is common to find one female and one male flower in an axil, with a solitary female flower in the opposite axil (Fig. 1A, M).The fruit wings are narrow and not easily noticeable until the fruit is fully mature and dry (Fig. 1C-E).The styles, which persist when the fruit is fully mature, are typically shorter than the fruit height (Fig. 1A-C).Mature fruits are blackish, 0.5-0.7 mm in length and 0.6-0.8mm in width (Fig. 1D, E).These traits also match the description of C. terrestris in recent taxonomic papers from Europe and South America (Hassemer and Lansdown 2018, Lansdown and Hassemer 2021).Callitriche turfosa Bertero ex Hegelmaier, which was previously referred to as subspecies Callitriche terrestris subsp.turfosa (Bertero ex Hegelm.)Bacigalupo and is reportedly very similar in morphology to C. terrestris, has a convex fruit surface (Lansdown and Hassemer 2021), whereas specimens KKmz1, KHsm1 and IbTcu1 have a relatively flat surface.Therefore, they were identified as C. terrestris rather than C. turfosa.
The other type of specimens showed a similar shoot appearance, but had different flower and fruit morphologies.These plants often produced fruits with elongated pedicels (>1 mm) usually directed downwards (Fig. 1G, H) and short pedicels were sometimes observed (Fig. 1K, L).Male and female flowers were often found on the same axil, whereas the opposite axil often lacked a flower (Fig. 1F, H, M).Mature fruits had clear wings on all sides (Fig. 1I, J).Styles were either erect or weakly recurved and persisted on fully mature fruits and were often longer than the fruit height (Fig. 1G, L).Mature fruits were brown, measuring 0.5-0.7 mm in length and 0.7-0.9mm in width (Fig. 1I).These characteristics were consistent with the keys for C. deflexa in recent literature (Lansdown et al. 2017a, Hassemer and Lansdown 2018, Lansdown and Hassemer 2021), leading us to conclude that these specimens were C. deflexa.
The shoots of both C. terrestris and C. deflexa are similar in appearance, but some differences become apparent when they are grown under the same conditions (Fig. 2).For example, the stem of C. deflexa tends to be longer and the mature leaves are rounder in shape when compared with C. terrestris (Fig. 2).These differences in shoot traits are stable when the plants are grown in a controlled environment, such as a growth chamber, but they may vary in different natural environments and are, thus, not suitable for rigid identification in the field.Previous studies have shown that the stomatal development of C. deflexa (denoted as C. terrestris in these papers) has some specific characteristics, such as amplifying division of stomatal lineage (Doll et al. 2021b) and stomata that differentiate on both sides of the leaf (amphistomy) (Doll et al. 2021a).Here, we confirmed that these characteristics were also shared by genuine C. terrestris (Fig. 2E, F).
Although the collected specimens showed differences in the traits described above, these traits were the keys to identifying C. deflexa and no traits could be used to identify C. terrestris.For example, flowering nodes in which a female flower is opposed by another female flower are also often observed in C. deflexa (Fig. 1G, K, M).Furthermore, the pedicel length of C. deflexa is variable and sometimes as short as that of C. terrestris (Fig. 1L).Thus, multiple traits of many flowers and fruits need to be comprehensively analysed for morphological identification.

DNA sequence analysis
Next, we analysed DNA sequences to evaluate the genetic divergence between the two specimen types.According to previous studies that extensively examined the phylogenetic relationships of the genus Callitriche ( Ito et al. 2017), matK and rbcL sequences were sequenced from the specimens.All sequences from specimens identified as C. deflexa on the basis of morphology were identical to C. deflexa analysed by Ito et al. (2017) (in the paper, this specimen was denoted as Callitriche compressa N.E.Br. on the basis of the original identification, but the identification of the specimen was corrected to C. deflexa by Lansdown et al. ( 2017a)) (Tables 2, 3).In contrast, the rbcL sequence of a specimen assigned to C. deflexa by Philbrick and Les (2000) (Table 3) did not match the rbcL sequence of C. deflexa.As some traits of the specimen used by Philbrick and Les (2000) are inconsistent with the above-mentioned description, it is highly likely that the specimen is not C. deflexa, but a different species from the current taxonomic point of view.All three specimens morphologically identified as C. terrestris shared identical sequences, with 16 variant sites in matK and four variant sites in rbcL when compared with C. deflexa (Table 2 and Table 3).The rbcL sequence was identical to that of C. terrestris analysed by Philbrick and Les (2000).
As the phylogenetic position of C. terrestris was not examined even in previous broad phylogenetic analyses (Ito et al. 2017, Prančl et al. 2020), we performed a phylogenetic tree reconstruction by combining matK and rbcL sequences (Fig. 3).The result placed C. terrestris in clade VI, according to Ito et al. (2017).It is not a sister group to the morphologically similar C. turfosa and the results do not support the systematics that placed the two taxa as subspecies.However, the validity of the identification of specimens whose sequences were analysed as C. turfosa must be reviewed using the current taxonomic view.Callitriche deflexa belonged to clade VII, which is consistent with the results of previous studies (Ito et al. 2017, Lansdown et al. 2017a).

Re-examination of the herbarium specimens
We re-examined herbarium specimens from KPM, which has the largest collection of specimens previously identified as C. terrestris in Japan.We confirmed that most of the herbarium specimens were C. terrestris, but some specimens showed the characteristics of C. deflexa: extensive variation in pedicel length, sometimes reaching more than 3 mm; clear wings on fruit edges (Fig. 4).This type of plant has been recorded in Kanagawa, Mie, Osaka and Okinawa Prefectures.The oldest specimen was collected in 1999 in Okinawa Prefecture, indicating that C. deflexa has been misidentified as C. terrestris in Japan for at least 24 years.
We also tried to amplify and sequence plastid DNA from some herbarium specimens deposited in KPM (Suppl.material 1).Due to the difficulties in DNA extraction from herbarium specimens, we could obtain only short fragments of matK sequences from limited specimens (Suppl.material 1).Nevertheless, we confirmed clear genotypic differences that were consistent with the morphological differences.All examined voucher specimens of NA1004372, NA1104918 and NA0112762; KPM and additional recentlycollected specimens (NA0162807 and NA0216556; KPM) had the same sequences as C. terrestris, which we confirmed using the freshly-collected specimens.We also succeeded in obtaining amplicons from three out of four specimens that are probably C. deflexa on the basis of the morphological traits and found that they have sequences distinct from those of C. terrestris.Except for one single nucleotide variant (SNV) site (position 562) found in the specimens from Mie and Kanagawa, the sequences were identical to that of C. deflexa collected in this study (Table 2).In conclusion, on the basis of both morphology and genetics, two clearly different types of specimens were found in this study: C. terrestris, as identified previously and C. deflexa.or UFboot support < 95% were omitted as they were not reasonably supported.Clade names (I to IX) were assigned according to Ito et al. (2017).Due to there being no sequence differences amongst the specimens obtained for this study, we used the sequence of only one specimen for each species.

Discussion
In this study, we found that two terrestrial Callitriche species have become naturalised in Japan: C. terrestris and C. deflexa.We propose the Japanese name "Nagae-awagoke" for C. deflexa, which means "terrestrial water-starwort with long pedicels," on the basis of its morphological features.This is the first report of C. deflexa in Japan because it has often been misidentified as C. terrestris in the past.In fact, a book on naturalised plants in Japan has a page for C. terrestris with photos of plants that resemble C. deflexa, as well as true C. terrestris ( Uemura et al. 2015).Additionally, plants that have been reported as C. terrestris in Ehime Prefecture exhibited the specific traits of C. deflexa ( Fukuoka and Hayakawa 2016).These misidentifications can be partially attributed to the difficulty in First records of non-native species Callitriche deflexa (Plantaginaceae), ...
distinguishing between the two species on the basis of their morphological traits.All the key traits observed in this study can be plastic in C. deflexa.Therefore, depending on the plant condition and number of observations, one may be unable to find any characteristic traits even from an actual C. deflexa specimen.For proper identification of C. deflexa and C. terrestris, it is necessary to observe as many flowers and fruits as possible from a single plant to determine whether it has plastic traits expected in C. deflexa.If it is not possible to observe a specimen with a sufficient number of flowers or fruits, it is recommended to allow the specimen to grow for a while or use DNA sequencing to determine the species.
The distribution of C. terrestris and C. deflexa in Japan needs to be investigated in the future.However, C. terrestris is found in only limited areas of the Kanto Region (Kanagawa and Ibaraki Prefectures), whereas C. deflexa is widely found in Kanto and western Japan.
Both species have been found in Kanagawa and Ibaraki Prefectures, so their distribution seems to overlap in the Kanto Region.Callitriche deflexa was once reported in Taiwan in the 1970s (Hsu and Yang 1976, Yang and Hsu 1998, Chen et al. 2013) and it was first collected from Okinawa, Japan, in 1999, suggesting that it may have colonised Japan from the southern region.Callitriche deflexa may have colonised multiple times, as the specimens examined in this study showed genetic variation in the matK sequence (Table 3).
The results of this study suggest ecological differences between the two species.In Japan, C. terrestris plants were found only from April to June (Katsuyama and Kawasumi 1999, Suppl.material 1).In fact, in the habitat in Ibaraki Prefecture, C. terrestris was not present in February, but was thriving in May and it then disappeared in August (Table 1).In contrast, C. deflexa was often collected in winter (Table 1), indicating that it is cold-tolerant and able to overwinter in Japan.Additionally, C. deflexa was found in August and September in Japan (Table 1), suggesting that it is tolerant to high summer temperatures.Therefore, the differences in the life cycles of C. deflexa and C. terrestris in Japan may contribute to their different dispersal patterns.
This study revealed that the plant specimens identified as C. terrestris in previous developmental studies were, in fact, C. deflexa (Koga et al. 2021, Doll et al. 2021b, Doll et al. 2021a).In a previous study, we analysed plasticity in leaf development (heterophylly) by using C. palustris and chose " C. terrestris" as a phylogenetically close, but nonheterophyllous relative for comparison of leaf development (Koga et al. 2021).Although the material was found to be C. deflexa in this study, C. deflexa is also a suitable, albeit not optimal, terrestrial non-heterophyllous plant.We also used C. deflexa as "C.terrestris" for studies of stomatal development and evolution in Callitriche (Doll et al. 2021a, Doll et al. 2021b).However, in the present study, we confirmed that C. terrestris exhibits stomatal development characteristics similar to those of C. deflexa: stomata differentiate on both sides of the leaf and stomatal lineages may undergo amplifying division (Fig. 2).It is notable that these features were confirmed in a terrestrial species of Callitriche that belongs to a clade different from previously examined C. deflexa and C. japonica.Thus, the conclusions of previous studies were not invalidated by the misidentification.

Figure 1 .
Figure 1.Flower and fruit morphologies of C. terrestris and C. deflexa.(A-E) Callitriche terrestris (KKmz1 and IbTcu1) and (F-L) C. deflexa (HN2 and FF2).A A node with two young female flowers and one male flower; B An immature fruit with a short deflexed pedicel; C A node with mature fruits; D, I Fully matured schizocarp abscised from the pedicel; E, J Mericarps; F A node with one young female flower and one male flower in an axil; G Young female flowers with elongated pedicels; H Mature fruit with an elongated pedicel; K A node with two female flowers and one male flower; L Mature fruits with short pedicels; M The proportion of flowering patterns of nodes.Scale bars: 1 mm.

Figure 2 .
Figure 2. Shoot morphologies of C. terrestris and C. deflexa.A A whole-plant image of C. terrestris; B A whole plant image of C. deflexa; C Images of shoots; D Leaf shapes; E Abaxial epidermis of C. terrestris leaf.Guard cells indicated by an arrowhead are surrounded by three cells of heterogeneous size, suggesting that this lineage has undergone amplifying division; F Stomatal index (stomatal number per epidermal cell) on the adaxial and abaxial sides of C. terrestris leaves (n = 5 each).Circles indicate the values of each leaf and crosses indicate mean values.Scale bars: A-C 1 cm, D 1 mm, E 100 µm.

Figure 3 .
Figure 3.Phylogenetic tree of a plastid gene dataset (matK and rbcL).A Maximum Likelihood tree reconstructed by concatenated alignment of matK and rbcL DNA sequences.Node values represent Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) support (%)/ultrafast bootstrap (UFboot) support (%).Node values with either SH-aLRT support < 80% or UFboot support < 95% were omitted as they were not reasonably supported.Clade names (I to IX) were assigned according toIto et al. (2017).Due to there being no sequence differences amongst the specimens obtained for this study, we used the sequence of only one specimen for each species.

Figure 4 .
Figure 4. Herbarium specimens of C. deflexa.A representative specimen with a magnified image of the female flowers with elongated pedicels.A M. Matsumoto NA0116625 (KPM) from Okinawa Pref.; B M. Matsumoto NA0203540 (KPM) from Mie Pref.; C M. Matsumoto NA0208310 (KPM) from Kanagawa Pref.Scale bars: 1 cm for the whole plant panels and 1 mm for the magnified panels.
6Ovate leaf venation simple; fruits clearly winged at the apex only or the wing at the apex distinctly wider than below or fruits unwinged; when fruit unwinged, bracts as long and wide as ripe fruits C. palustris