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. 2024 Jan 15;12:e115142. doi: 10.3897/BDJ.12.e115142

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

Hiroyuki Koga 1,✉, Yuki Doll 1, Wataru Ohnishi 2, Hirokazu Tsukaya 1
PMCID: PMC10837795  PMID: 38314126

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.

Keywords: Callitrichedeflexa , Callitricheterrestris , non-native species, new record, Japan

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: Callitrichehermaphroditica L., Callitrichejaponica Engelm. ex Hegelmaier, Callitrichepalustris L. and Callitrichefuscicarpa Lansdown (Lansdown 2006b). In addition, Callitricheterrestris Muhl. ex Raf. (Katsuyama and Kawasumi 1999) and Callitrichestagnalis Scop. (Morita and Lee 1998) have been reported as naturalised species (Uemura et al. 2015). Callitricheterrestris 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). Callitrichestagnalis 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 in Japan have been conducted by us (Doll et al. 2021a, Doll et al. 2021b, Koga et al. 2021). To date, only C.japonica and C.terrestris have been recognised as terrestrial Callitriche species in Japan. Katsuyama and Kawasumi (1999) distinguished C.terrestris from C.japonica on the basis of its spatulate leaf shape, presence of pedicels and less pronounced fruit wings. According to these key traits, terrestrial Callitriche specimens from various regions of Japan, including those used in our studies, were identified as C.terrestris. However, laboratory cultivation and observations have shown us that at least the specimens used in our experiments had morphological traits that differed slightly from those originally described by Katsuyama and Kawasumi (1999). Therefore, we conducted a taxonomic re-examination of the plants regarded as C.terrestris by collecting them from several localities in Japan as well as observing herbarium specimens. Our investigations revealed Callitrichedeflexa A. Braun as a previously misidentified and non-native terrestrial species in Japan.

Materials and methods

Plant collection and morphological observations

Terrestrial Callitriche specimens were collected from various locations, such as Hyogo (HN2), Osaka (OH1), Fukuoka (FF2), Ibaraki (IbTkb1 and IbTcu1), Tokyo (TKoi1) and Kanagawa (KKmz1, KHd1, KHsm1) Prefectures (Table 1). The plant strain from Hyogo Prefecture (HN2) has been previously used in developmental biology studies (Doll et al. 2021a, Doll et al. 2021b, Koga et al. 2021). Some specimens co-occurred with C.japonica, but they could be easily distinguished by their shoot forms and fruit morphologies (Katsuyama 2018). The localities of KKmz1 and KHsm1 in Kanagawa Prefecture matched with the information of voucher specimens for C.terrestris, M. Matsumoto NA0112762 (KPM) and C. Hasekura NA0112764 (KPM), respectively (Katsuyama and Kawasumi 1999).

Table 1.

Plants collected for this study

Name Species Location Collected month Habitat Specimen ID Notes
HN2 C.deflexa Nishinomiya, Hyogo Sep 2015 Farm 00256641(TI) Used in previous studies
(Doll et al. 2021a, Doll et al. 2021b, Koga et al. 2021),
5th generation plant of inbred line was examined
OH1 C.deflexa Hirakata, Osaka Sep 2015 Farm 00256643(TI) 1st generation plant of inbred line was examined
FF2 C.deflexa Fukuoka, Fukuoka Feb 2020 Plantation 00256647(TI) 1st generation plant of inbred line was examined
IbTkb1 C.deflexa Tsukuba, Ibaraki Feb 2022 Green house 00256639(TI)
KHd1 C.deflexa Hodogaya, Kanagawa Mar 2022 Plantation 00256640(TI) Also found in Aug 2022
TKoi1 C.deflexa Koishikawa, Tokyo Mar 2022 Plantation 00256646(TI)
KKmz1 C.terrestris Kamimizo, Kanagawa May 2022 Playground 00256644(TI) Locality consistent with NA0112762(KPM), not found in Aug 2022
KHsm1 C.terrestris Hashimoto, Kanagawa May 2022 Garden 00256645(TI) Locality consistent with NA0112764(KPM), not found in Aug 2022
IbTcu1 C.terrestris Tsuchiura, Ibaraki May 2022 Park 00256642(TI) Not found in Mar and Aug 2022
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To observe the flower and fruit morphologies, the collected plants were cultured in soil (Mizukusa-ichiban sand, GEX, Japan) in a growth chamber with a long-day condition (16 h light and 8 h dark) at 22˚C. Some specimens were plants that were inbred for 1–5 generations after collection. The voucher specimens were deposited to Herbarium of the Department of Botany, University of Tokyo (TI; Table 1). Herbarium collections of Kanagawa Prefectural Museum of Natural History (KPM) were also examined for both morphological and genetic investigations (Suppl. material 1).

DNA extraction and amplification of barcoding regions

To obtain genomic DNA for analysis, we extracted DNA from the shoot tips of herbarium specimens and living plants. For herbarium specimens, we first homogenised the dry samples by using TissueLyser II (Qiagen, Hilden, Germany) with zirconia beads and then performed DNA extraction by using the cetyl trimethyl ammonium bromide method, followed by purification with AMPure XP beads (Beckman Coulter, Brea, California, USA). For fresh samples, we used a DNeasy Plant Mini Kit (Qiagen). We amplified matK and rbcL regions of the extracted DNA by using PCR with specific primer sets: matK_390_F (5′-CGATCTATTCATTCAATATTTC-3′) and matK_1326R (5′-TCTAGCACACGAAAGTCGAAGT-3′) (Cuénoud et al. 2002); rbcL_5F (5′-ACCACAAACAGARACTAAAGC-3′) and rbcL_1210R (5′-AAGGRTGYCCTAAAGTTCCTCC-3′) (Les et al. 1993, Philbrick and Les 2000). In cases where PCR with the primer sets for fresh samples was unsuccessful for herbarium samples, we designed a new primer set that specifically targeted a short (199 bp) region of matK in the examined species: matK_497F (5′-ATTGGGTAAAAGATGCCTCTTCTTTGC-3′) and matK_695R (5′-TGAGAAGATTGGTTACGTAGAAAGACG-3′). We performed PCR with PrimeSTAR GXL Polymerase (Takara Bio, Shiga, Japan) or Ex Premier Polymerase (Takara Bio) and purified the PCR products with AMPure XP beads (Beckman Coulter). Sanger sequencing was used to sequence the purified products with the primers used for PCR. The resulting sequences were deposited in NCBI/DDBJ/EBI (accession numbers listed in Table 2 and Table 3).

Table 2.

SNVs in rbcL.

Specimen Position in ORF Accession Source
159 165 504 555 657 793 1072 1168
LC177674.1 C.deflexa A T C C G A A T LC177674 Ito et al. (2017)
FF2 . . . . . . . . LC765361
HN2 . . . . . . . . LC765362
KHd1 . . . . . . . . LC765365
OH1 . . . . . . . . LC765368
TKoi1 . . . . . . . . LC765369
IbTkb1 . . . . . . . . LC765364
KHsm1 . . . T A . C C LC765366
KKmz1 . . . T A . C C LC765367
IbTcu1 . . . T A . C C LC765363
AF248024.1 C.terrestris . . . T A . C C AF248024 Philbrick and Les (2000)
AF248012.1 (C.deflexa*)
*likely misidentification		 G C T T . G C C AF248012 Philbrick and Les (2000)
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Table 3.

SNVs in matK.

Specimen Position in ORF Accession Source
448 562 564 586 598 599 600 625 661 699 876 966 1045 1098 1193 1250
LC176831.1 C.deflexa A A G G G A A T T A T A A T T C LC176831 Ito et al. (2017)
FF2 . . . . . . . . . . . . . . . . LC765346
HN2 . . . . . . . . . . . . . . . . LC765347
KHd1 . . . . . . . . . . . . . . . . LC765350
OH1 . . . . . . . . . . . . . . . . LC765353
TKoi1 . . . . . . . . . . . . . . . . LC765354
IbTkb1 . . . . . . . . . . . . . . . . LC765349
KHsm1 T T A T T T C G C G A C T A G T LC765351
KKmz1 T T A T T T C G C G A C T A G T LC765352
IbTcu1 T T A T T T C G C G A C T A G T LC765348
NA0208310 (KPM) NA T . . . . . . . NA NA NA NA NA NA NA LC765676
NA0203540 (KPM) NA T . . . . . . . NA NA NA NA NA NA NA LC765677
NA0202813 (KPM) . . . . . . . . . . . . . . . . LC765359
NA1004372 (KPM) NA T A T T T C G C NA NA NA NA NA NA NA LC765355
NA1104918 (KPM) NA T A T T T C G C NA NA NA NA NA NA NA LC765356
NA0112762 (KPM) NA T A T T T C G C NA NA NA NA NA NA NA LC765357
NA0162807 (KPM) NA T A T T T C G C NA NA NA NA NA NA NA LC765360
NA0216556 (KPM) NA T A T T T C G C NA NA NA NA NA NA NA LC765358
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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).

Taxon treatments

Callitriche deflexa

A. Braun, 1846

D7B8CE9C-F2DC-52CE-A403-7975D2715EB1

  • ナガエアワゴケ Nagae-awagoke (terrestrial water-starwort with long pedicels) (nov.); broad-fruited water-starwort (Lansdown 2008).

Materials

  1. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/2244423416; catalogNumber: KPM-NA0208310; individualCount: 1; occurrenceStatus: present; occurrenceID: F529E51B-CE6E-52A8-8735-4A134E3B505C; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Yamato City|Minamirinkan; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Yamato City; locality: Minamirinkan; decimalLatitude: 35.490755; decimalLongitude: 139.440551; geodeticDatum: WGS84; Event: eventDate: 2016-06-08T00:00:00; year: 2016; month: 6; day: 8; Record Level: type: text; modified: 2019-02-28T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  2. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/3324114555; catalogNumber: KPM-NA0203540; individualCount: 1; occurrenceStatus: present; occurrenceID: D34E21E0-9C75-5605-985C-19F62C0EDA26; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Mie Prefecture|Yokkaichi City; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Mie Prefecture; county: Yokkaichi City; decimalLatitude: 34.98294; decimalLongitude: 136.66162; geodeticDatum: WGS84; Event: eventDate: 2011-05-30T00:00:00; year: 2011; month: 5; day: 30; Record Level: type: text; modified: 2020-11-27T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  3. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/3324109531; catalogNumber: KPM-NA0202813; individualCount: 1; occurrenceStatus: present; occurrenceID: D9FE4257-7A60-5D82-968A-2A6D22810830; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Osaka Prefecture|Hannan City|Yamanakadani; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Osaka Prefecture; county: Hannan City; locality: Yamanakadani; decimalLatitude: 34.32601; decimalLongitude: 135.27158; geodeticDatum: WGS84; Event: eventDate: 2013-04-09T00:00:00; year: 2013; month: 4; day: 9; Record Level: type: text; modified: 2020-11-27T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  4. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/1829883645; catalogNumber: KPM-NA0116625; individualCount: 1; occurrenceStatus: present; occurrenceID: 9B6F284A-FE87-5EAC-8C63-3EACA2E427AC; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Okinawa Prefecture|Nago-shi; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Okinawa Prefecture; county: Nago-shi; decimalLatitude: 26.591944; decimalLongitude: 127.977222; geodeticDatum: WGS84; Event: eventDate: 1999-03-09T00:00:00; year: 1999; month: 3; day: 9; Record Level: type: text; modified: 2013-07-01T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  5. Type status: Other material. Occurrence: catalogNumber: TI00256641; occurrenceID: D2A453DF-5FC9-53BC-BB0B-8D95B7D2A34B; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Hyogo; county: Nishinomiya-shi; decimalLatitude: 34.7695; decimalLongitude: 135.3653; geodeticDatum: WGS84; Event: eventDate: 2015-09-27T00:00:00; year: 2015; month: 9; day: 27; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

  6. Type status: Other material. Occurrence: catalogNumber: TI00256643; recordedBy: Hiroyuki Koga; occurrenceID: 41DA7F8B-F3FC-5D4A-805F-91BA171BB6FE; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Osaka; county: Hirakata-shi; decimalLatitude: 34.8176; decimalLongitude: 135.6701; geodeticDatum: WGS84; Event: eventDate: 2015-09-20T00:00:00; year: 2015; month: 9; day: 20; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

  7. Type status: Other material. Occurrence: catalogNumber: TI00256647; recordedBy: Hiroyuki Koga; individualCount: 1; occurrenceID: 45915E75-EB4B-5FF1-9108-5C4EFB15EEB5; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Fukuoka; county: Fukuoka-shi; decimalLatitude: 33.5808; decimalLongitude: 130.3928; geodeticDatum: WGS84; Event: eventDate: 2020-02-02T00:00:00; year: 2020; month: 2; day: 2; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

  8. Type status: Other material. Occurrence: catalogNumber: TI00256639; recordedBy: Hiroyuki Koga; individualCount: 1; occurrenceID: A29E5A5F-72E2-5C91-B4E2-0D6D9769F623; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Ibaraki; county: Tsukuba-shi; decimalLatitude: 36.0625; decimalLongitude: 140.0799; geodeticDatum: WGS84; Event: eventDate: 2022-02-25T00:00:00; year: 2022; month: 2; day: 25; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

  9. Type status: Other material. Occurrence: catalogNumber: TI00256640; recordedBy: Hiroyuki Koga; individualCount: 1; occurrenceID: EBADE9B7-DA51-51A6-AE56-A6240EE2FA31; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Kanagawa; county: Yokohama-shi; municipality: Hodogaya-ku; decimalLatitude: 35.4440; decimalLongitude: 139.5969; geodeticDatum: WGS84; Event: eventDate: 2022-03-09T00:00:00; year: 2022; month: 3; day: 9; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

  10. Type status: Other material. Occurrence: catalogNumber: TI00256646; recordedBy: Yuki Doll; individualCount: 1; occurrenceID: F86F621B-9757-58B9-A678-CE63CF60553E; Taxon: scientificName: Callitrichedeflexa A. Braun; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: deflexa; taxonRank: species; vernacularName: ナガエアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Tokyo; county: Bunkyo-ku; municipality: Koishikawa; decimalLatitude: 35.7130; decimalLongitude: 139.7510; geodeticDatum: WGS84; Event: eventDate: 2022-03-06T00:00:00; year: 2022; month: 3; day: 6; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

Distribution

Native to Central South America: southern Brazil, Paraguay, Uruguay and northern Argentina (Fassett 1951, Lansdown and Hassemer 2021). Naturalised in Australia (Lansdown and Hassemer 2021, Lansdown 2022), Europe (Lansdown 2006a, Lansdown 2008, Raab-Straube 2014), Africa (Lansdown et al. 2017a, Lansdown et al. 2022), Taiwan (Chen et al. 2013) and Japan (newly reported here).

Callitriche terrestris

Muhl. ex Raf., 1808

21103395-D5AC-5E00-9074-FE2C85643ADB

Materials

  1. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/187978085; catalogNumber: KPM-NA1004382; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 9FE12147-770D-5906-9AD9-DBDBBA5C6169; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa|Ebina-shi|Kawaraguchi,Ebina City, Kawara-guti; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa; county: Ebina-shi; locality: Kawaraguchi,Ebina City, Kawara-guti; decimalLatitude: 35.449095; decimalLongitude: 139.378057; geodeticDatum: WGS84; Event: eventDate: 1984-06-11T00:00:00; year: 1984; month: 6; day: 11; Record Level: type: text; modified: 2007-05-09T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  2. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/187978079; catalogNumber: KPM-NA1004375; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: CB3D07CB-089A-51F4-AE93-A81FA3DB55E0; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa|Yokohama-shi Nishi-ku|Motokubo-cho,Yokohama City, Nishi-ku, Motokubo-cho; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa; county: Yokohama-shi Nishi-ku; locality: Motokubo-cho,Yokohama City, Nishi-ku, Motokubo-cho; decimalLatitude: 35.440766; decimalLongitude: 139.60304; geodeticDatum: WGS84; Event: eventDate: 1985-05-22T00:00:00; year: 1985; month: 5; day: 22; Record Level: type: text; modified: 2007-05-09T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  3. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/187978076; catalogNumber: KPM-NA1004372; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: D5778624-3727-5AC0-B429-9B19F1F920EB; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa|Yokohama-shi Kanagawa-ku|Yokohama City, Kanagawa-ku, Mitsuzawa Park; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa; county: Yokohama-shi Kanagawa-ku; locality: Yokohama City, Kanagawa-ku, Mitsuzawa Park; decimalLatitude: 35.474095; decimalLongitude: 139.603039; geodeticDatum: WGS84; Event: eventDate: 1985-07-04T00:00:00; year: 1985; month: 7; day: 4; Record Level: type: text; modified: 2007-05-09T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  4. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/388800331; catalogNumber: KPM-NA0112761; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 3832C2BA-8F0D-5052-AC76-C778571D0A67; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Yokohama-shi, Minami-ku; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Yokohama-shi, Minami-ku; decimalLatitude: 35.432434; decimalLongitude: 139.628038; geodeticDatum: WGS84; Event: eventDate: 1997-06-13T00:00:00; year: 1997; month: 6; day: 13; Record Level: type: text; modified: 2013-07-05T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  5. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/388800334; catalogNumber: KPM-NA0112764; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 3E59BC2D-4F3A-5AD8-B143-B2CA60D17C36; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Sagamihara-shi; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Sagamihara-shi; decimalLatitude: 35.607403; decimalLongitude: 139.340555; geodeticDatum: WGS84; Event: eventDate: 1998-06-18T00:00:00; year: 1998; month: 6; day: 18; Record Level: type: text; modified: 2013-07-05T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  6. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/388800332; catalogNumber: KPM-NA0112762; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: FA544454-3E6D-5B78-B8BF-6917C4F45006; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Sagamihara-shi; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Sagamihara-shi; decimalLatitude: 35.549079; decimalLongitude: 139.365555; geodeticDatum: WGS84; Event: eventDate: 1998-06-01T00:00:00; year: 1998; month: 6; day: 1; Record Level: type: text; modified: 2013-07-05T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  7. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/245530607; catalogNumber: KPM-NA1104918; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 24E5E4FE-575E-557B-A18E-D84585642D20; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Yokohama-shi, Hodogaya-ku; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Yokohama-shi, Hodogaya-ku; decimalLatitude: 35.449098; decimalLongitude: 139.590541; geodeticDatum: WGS84; Event: eventDate: 1992-06-27T00:00:00; year: 1992; month: 6; day: 27; Record Level: type: text; modified: 2010-06-09T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  8. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/2244423413; catalogNumber: KPM-NA0208327; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: CD68C85C-CE86-5966-AC57-5648D008283F; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa-Ken|Sagamihara-shi, Minami-ku|Shimomizo; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa-Ken; county: Sagamihara-shi, Minami-ku; locality: Shimomizo; decimalLatitude: 35.524084; decimalLongitude: 139.378055; geodeticDatum: WGS84; Event: eventDate: 2004-04-20T00:00:00; year: 2004; month: 4; day: 20; Record Level: type: text; modified: 2019-02-28T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  9. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/388800487; catalogNumber: KPM-NA0162807; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 84880289-CB0F-5ED8-AA84-0523BF49C19E; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Yokohama-shi, Izumi-ku; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Yokohama-shi, Izumi-ku; decimalLatitude: 35.390771; decimalLongitude: 139.49055; geodeticDatum: WGS84; Event: eventDate: 2009-07-07T00:00:00; year: 2009; month: 7; day: 7; Record Level: type: text; modified: 2013-07-06T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  10. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/388800461; catalogNumber: KPM-NA0136933; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 015992B6-44B1-591F-8044-5AD8086A4196; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Yokohama-shi, Minami-ku; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Yokohama-shi, Minami-ku; decimalLatitude: 35.432083; decimalLongitude: 139.576889; geodeticDatum: WGS84; Event: eventDate: 2009-07-07T00:00:00; year: 2009; month: 7; day: 7; Record Level: type: text; modified: 2013-07-05T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PRESERVED_SPECIMEN

  11. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/1829916404; catalogNumber: KPM-NA0176499; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 036F3363-A5A9-570E-993A-78A695F9BF97; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Hadano-shi|Minamiyana; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Hadano-shi; locality: Minamiyana; decimalLatitude: 35.365769; decimalLongitude: 139.265567; geodeticDatum: WGS84; Event: eventDate: 2014-06-08T00:00:00; year: 2014; month: 6; day: 8; Record Level: type: text; modified: 2015-12-19T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  12. Type status: Other material. Occurrence: occurrenceDetails: http://api.gbif.org/v1/occurrence/2244425949; catalogNumber: KPM-NA0216556; individualCount: 1; occurrenceStatus: PRESENT; occurrenceID: 52C34102-9BAC-54BE-BCEA-A32688FA0A4A; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: higherGeography: Japan|Kanagawa Prefecture|Yokohama City, Hodogaya-ku|Hoshikawa; continent: ASIA; country: Japan; countryCode: JP; stateProvince: Kanagawa Prefecture; county: Yokohama City, Hodogaya-ku; locality: Hoshikawa; decimalLatitude: 35.457431; decimalLongitude: 139.590541; geodeticDatum: WGS84; Event: eventDate: 2015-06-02T00:00:00; year: 2015; month: 6; day: 2; Record Level: type: text; modified: 2019-02-28T00:00:00.000+00:00; language: en; institutionID: institutionID; collectionID: collectionID; institutionCode: KPM; collectionCode: NA; basisOfRecord: PreservedSpecimen

  13. Type status: Other material. Occurrence: catalogNumber: TI00256644; recordedBy: Hiroyuki Koga; occurrenceID: C1754382-903E-52EE-90E8-46C7E3A26881; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Kanagawa; county: Sagamihara-shi; municipality: Kamimizo; decimalLatitude: 35.548853; decimalLongitude: 139.370462; geodeticDatum: WGS84; Event: eventDate: 2022-05-02T00:00:00; year: 2022; month: 5; day: 2; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

  14. Type status: Other material. Occurrence: catalogNumber: TI00256645; recordedBy: Hiroyuki Koga; occurrenceID: D6172080-FA45-59E8-949D-DD1AB9A222EB; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Kanagawa; county: Sagamihara-shi; municipality: Hoshimoto; decimalLatitude: 35.601606; decimalLongitude: 139.347401; geodeticDatum: WGS84; Event: eventDate: 2022-05-02T00:00:00; year: 2022; month: 5; day: 2; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

  15. Type status: Other material. Occurrence: catalogNumber: TI00256642; recordedBy: Hiroyuki Koga; occurrenceID: 4E8227EB-4953-538E-8541-0194F6E6CC47; Taxon: scientificName: Callitricheterrestris Muhl. ex Raf.; kingdom: Plantae; phylum: Tracheophyta; class: Magnoliopsida; order: Lamiales; family: Plantaginaceae; genus: Callitriche; specificEpithet: terrestris; taxonRank: species; vernacularName: アメリカアワゴケ; taxonomicStatus: accepted; Location: continent: Asia; country: Japan; countryCode: JP; stateProvince: Ibaraki; county: Tsuchiura-shi; locality: Ottonuma Park; decimalLatitude: 36.04474; decimalLongitude: 140.15070; geodeticDatum: WGS84; Event: eventDate: 2022-05-20T00:00:00; year: 2022; month: 5; day: 20; Record Level: language: en; institutionCode: TI; basisOfRecord: PreservedSpecimen

Distribution

Native to North and South America: United States, Mexico and South American countries (Fassett 1951, Lansdown 2009, Hassemer and Lansdown 2018, Lansdown and Hassemer 2021). Naturalised in Europe (Lansdown 2006a, Lansdown 2008, Lansdown et al. 2017b), Taiwan and Japan (Katsuyama and Kawasumi 1999).

Identification Keys

Keys to identification of Callitriche species recorded from Japan

1 Bracts absent 2
– Bract present 5
2 Ovate or spatulated leaves absent. Linear submerged leaves present C.hermaphroditica
– Ovate or spatulated leaves present. Linear submerged leaves absent 3
3 Fruits narrower at base than at apex; fruits subsessile C.japonica
– Base of fruit not narrowed; ripe fruits clearly pedicellate > 0.5 mm 4
4 Some pedicels > 1 mm; some nodes in which female and male flowers in one axil opposed by a non-flowering axil; ripe fruits clearly winged; some styles > 1 mm in length C.deflexa
– All pedicels ≤ 1 mm; when female and male flowers in a leaf axil, always more than one female flower and/or one male flower in the opposite axil; fruit wing unclear; style always ≤ 1 mm long, shorter than ripe fruit C.terrestris
5 Fruits almost the same length and width, with wings on all edges C.stagnalis
– Fruits longer than wide 6
6 Ovate 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
– Ovate leaf venation complex; fruits unwinged or only narrowly winged all around; bracts as long as, but narrower than ripe fruitsOvate leaf venation complex; fruits unwinged or only narrowly winged all around; bracts as long as, but narrower than ripe fruits C.fuscicarpa
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We provide identification keys for the species level. See Lansdown (2006b) for varieties of C.palustris.

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 0.6–0.8 mm 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). Callitricheturfosa Bertero ex Hegelmaier, which was previously referred to as subspecies Callitricheterrestrissubsp.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.

Figure 1.

Figure 1.

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Flower and fruit morphologies of C.terrestris and C.deflexa. (A–E) Callitricheterrestris (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.

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 0.7–0.9 mm 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).

Figure 2.

Figure 2.

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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.

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 Callitrichecompressa 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. Callitrichedeflexa belonged to clade VII, which is consistent with the results of previous studies (Ito et al. 2017, Lansdown et al. 2017a).

Figure 3.

Figure 3.

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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 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.

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.

Figure 4.

Figure 4.

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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.

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 recently-collected 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.

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 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. Callitrichedeflexa 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. Callitrichedeflexa 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 non-heterophyllous 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.

Supplementary Material

XML Treatment for Callitriche deflexa
XML Treatment for Callitriche terrestris
Supplementary material 1

Species occurrences

Hiroyuki Koga, Yuki Doll, Wataru Ohnishi, Horokazu Tsukaya

Data type

Table

Brief description

A list of examined herbarium samples.

File: oo_930177.xlsx

bdj-12-e115142-s001.xlsx (13.1KB, xlsx)
Supplementary material 2

Accession numbers of sequences used in phylogenetic analysis

Hiroyuki Koga, Yuki Doll, Wataru Ohnishi, Horokazu Tsukaya

Data type

Table

Brief description

Sequence information of the phylogenetic analysis.

File: oo_930178.xlsx

bdj-12-e115142-s002.xlsx (13.6KB, xlsx)

Acknowledgements

We thank Masumi Murase, Masayuki Kimura, Naruhisa Matsuoka, Naoto Kokubun, Teruo Katsuyama and Yasuro Kadono for providing information on the collection sites of the specimens. This study was supported by a Grant-in-Aid for Early-Career Scientists to HK (JP20K15816; JSPS), a Grant-in-Aid for Scientific Research (C) to WO (JP21K01012; JSPS) and a Grant-in-Aid for Scientific Research on Innovation Areas to HT (JP19H05672; MEXT).

References

  1. Bean A. R. A taxonomic revision of Callitriche L. (Callitrichaceae) in Australia. Austrobaileya: A Journal of Plant Systematics. 2007;7(3):545–554. doi: 10.5962/p.299754. [DOI] [Google Scholar]
  2. Chen C. F., Lin C. W., Yang S. Z. Callitrichedeflexa A. Braun ex Hegelm., reconfirmed as a naturalized plant in Taiwan. Taiwan Journal of Forest Science. 2013;28:153–158. [Google Scholar]
  3. Cuénoud Philippe, Savolainen Vincent, Chatrou Lars W., Powell Martyn, Grayer Renée J., Chase Mark W. Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. American Journal of Botany. 2002;89(1):132–144. doi: 10.3732/ajb.89.1.132. [DOI] [PubMed] [Google Scholar]
  4. Deschamp P. A., Cooke T. J. Leaf dimorphism in the aquatic angiosperm Callitricheheterophylla. American Journal of Botany. 1985;72:1377. doi: 10.2307/2443509. [DOI] [Google Scholar]
  5. Doll Yuki, Koga Hiroyuki, Tsukaya Hirokazu. Callitriche as a potential model system for evolutionary studies on the dorsiventral distribution of stomata. Plant Signaling & Behavior. 2021;16(11):1978201. doi: 10.1080/15592324.2021.1978201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Doll Yuki, Koga Hiroyuki, Tsukaya Hirokazu. The diversity of stomatal development regulation in Callitriche is related to the intrageneric diversity in lifestyles. Proceedings of the National Academy of Sciences. 2021;118(14):e202635111. doi: 10.1073/pnas.2026351118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Erbar C., Leins P. In: Flowering plants - Dicotyledons. The families and genera of vascular plants. Kubitzki Klaus, Rohwer Jens G., Bittrich Volker., editors. Vol. 7. Springer; Berlin: 2004. Callitrichaceae.50–56. [DOI] [Google Scholar]
  8. Fassett N. C. Callitriche in the new world. Rhodora. 1951;53:161. doi: 10.2307/23305272. [DOI] [Google Scholar]
  9. Fukuoka G., Hayakawa H. New naturalization of Callitricheterrestris in Ehime Prefecture, Japan. Ehimeayame. 2016;47 [Google Scholar]
  10. Hassemer Gustavo, Lansdown Richard V. Notes on the genus Callitriche (Plantaginaceae, Callitricheae) in South America, and an identification key for Brazil. Webbia. 2018;73(1):55–61. doi: 10.1080/00837792.2018.1443552. [DOI] [Google Scholar]
  11. Hoang Diep Thi, Chernomor Olga, von Haeseler Arndt, Minh Bui Quang, Vinh Le Sy. UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution. 2017;35(2):518–522. doi: 10.1093/molbev/msx281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hsu K. H., Yang Y. P. In: Flora of Taiwan. Committee Flora of Tiwan Editorial., editor. Epoch Pub. Co; Taipei: 1976. Callitrichaceae.436–438 [Google Scholar]
  13. Ito Yu, Tanaka Norio, Barfod Anders S., Kaul Robert B., Muasya A. Muthama, Garcia-Murillo Pablo, De Vere Natasha, Duyfjes Brigitta E. E., Albach Dirk C. From terrestrial to aquatic habitats and back again: molecular insights into the evolution and phylogeny of Callitriche (Plantaginaceae) Botanical Journal of the Linnean Society. 2017;184(1):46–58. doi: 10.1093/botlinnean/box012. [DOI] [Google Scholar]
  14. Jones Herbert. Heterophylly in some species of Callitriche, with especial reference to Callitricheintermedia. Annals of Botany. 1955;19(2):226–245. doi: 10.1093/oxfordjournals.aob.a083426. [DOI] [Google Scholar]
  15. Katoh K., Standley D. M. MAFFT Multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution. 2013;30(4):772–780. doi: 10.1093/molbev/mst010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Katsuyama T., Kawasumi E. Callitricheterrestris Raf. newly naturalized in Japan. Journal of Japanese Botany. 1999;74:181–182. Japanese. [Google Scholar]
  17. Katsuyama T. In: Kanagawaken shokubutsushi 2018 (Flora of Kanawaga 2018) Association Flora-Kanagawa., editor. Flora-Kanagawa Association; Odawara: 2018. Callitriche L.1388-1389. Japanese. [Google Scholar]
  18. Koga Hiroyuki, Doll Yuki, Hashimoto Kei, Toyooka Kiminori, Tsukaya Hirokazu. Dimorphic leaf development of the aquatic plant Callitrichepalustris L. through differential cell division and expansion. Frontiers in Plant Science. 2020;11:269. doi: 10.3389/fpls.2020.00269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koga Hiroyuki, Kojima Mikiko, Takebayashi Yumiko, Sakakibara Hitoshi, Tsukaya Hirokazu. Identification of the unique molecular framework of heterophylly in the amphibious plant Callitrichepalustris L. The Plant Cell. 2021;33(10):3272–3292. doi: 10.1093/plcell/koab192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lansdown Richard V. The genus Callitriche (Plantaginaceae, Callitricheae) in Australasia and Oceania. Phytotaxa. 2022;547(3):243–284. doi: 10.11646/phytotaxa.547.3.3. [DOI] [Google Scholar]
  21. Lansdown Richard V., Rhazi Laila, Saber Er-Riyahi, Madihi Mohammed El, Grillas Patrick. The genus Callitriche (Plantaginaceae, Callitricheae) in Morocco. Phytotaxa. 2022;555(1):53–72. doi: 10.11646/phytotaxa.555.1.4. [DOI] [Google Scholar]
  22. Lansdown R. V. Notes on the water-starworts (Callitriche) recorded in Europe. Watsonia. 2006;26:105–120. [Google Scholar]
  23. Lansdown R. V. The genus Callitriche (Callitrichaceae) in Asia. Novon: A Journal for Botanical Nomenclature. 2006;16:354–361. doi: 10.3417/1055-3177(2006)16. [DOI] [Google Scholar]
  24. Lansdown R. V. Water-starworts: Callitriche of Europe. Botanical Society of the British Isles; London: 2008. 180 [Google Scholar]
  25. Lansdown R. V. Nomenclatural Notes on Callitriche (Callitrichaceae) in North America. Novon: A Journal for Botanical Nomenclature. 2009;19:364–369. doi: 10.3417/2008030. [DOI] [Google Scholar]
  26. Lansdown R. V., Glen R., Hassemer G. Notes on the genus Callitriche (Plantaginaceae) in South Africa. Phytotaxa. 2017;317:219–225. doi: 10.11646/phytotaxa.317.3.6. [DOI] [Google Scholar]
  27. Lansdown R. V., Bazos I., Caria M. C., Troia A., Wieringa J. J. New distribution and taxonomic information on Callitriche (Plantaginaceae) in the Mediterranean region. Phytotaxa. 2017;313:91–104. doi: 10.11646/phytotaxa.313.1.6. [DOI] [Google Scholar]
  28. Lansdown R. V., Hassemer G. The genus Callitriche (Plantaginaceae: Callitricheae) in South America. Phytotaxa. 2021;501:85–118. doi: 10.11646/phytotaxa.501.1.3. [DOI] [Google Scholar]
  29. Les D., Garvin D., Wimpee C. Phylogenetic studies in the monocot subclass Alismatidae: evidence for a reappraisal of the aquatic order Najadales. Molecular Phylogenetics and Evolution. 1993;2:304–314. doi: 10.1006/mpev.1993.1029. [DOI] [PubMed] [Google Scholar]
  30. Minh Bui Quang, Schmidt Heiko A, Chernomor Olga, Schrempf Dominik, Woodhams Michael D, von Haeseler Arndt, Lanfear Robert. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Molecular biology and evolution. 2020;37(5):1530–1534. doi: 10.1093/molbev/msaa015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morita H., Lee D-J. Callitrichestagnalis Scop. (Callitrichaceae) occurring in water cress fields in Yamanashi prefecture, Japan. Jouranl of Japanease Botany. 1998;73:48–50. [Google Scholar]
  32. Philbrick C. T., Jansen R. K. Phylogenetic Studies of North American Callitriche (Callitrichaceae) Using Chloroplast DNA Restriction Fragment Analysis. Systematic Botany. 1991;16:478. doi: 10.2307/2419338. [DOI] [Google Scholar]
  33. Philbrick C. T., Les D. H. Phylogenetic studies in Callitriche: mplications for interpretation of ecological, karyological and pollination system evolution. Aquatic Botany. 2000;68:123–141. doi: 10.1016/s0304-3770(00)00114-5. [DOI] [Google Scholar]
  34. Prančl J, Fehrer J., Caklová P., Bambasová V., Lučanová M., Kaplan Z. Intricate evolutionary history of Callitriche (Plantaginaceae) taxa elucidated by a combination of DNA sequencing and genome size. TAXON. 2020;69:1016–1041. doi: 10.1002/tax.12315. [DOI] [Google Scholar]
  35. Raab-Straube E. V. Euro Med-Checklist Notulae, 3. Willdenowia. 2014;44:287–299. doi: 10.3372/wi.44.44211. [DOI] [Google Scholar]
  36. Uemura S., Katsuyama T., Shimizu N., Mizuta M., Morita H., Hirota N., Ikehara N. Nihon Kika Shokubutsu Shashin Zukan Dai 2 Kan. Zenkoku Nourin Kyouiku Kyokai; Tokyo: 2015. 194-195. Japanese. [Google Scholar]
  37. Yang Y. P., Hsu K. In: Flora of Taiwan. 2. Committee Flora of Taiwan Editorial., editor. Vol. 4. Editorial Committee of the Flora of Taiwan; Taipei: 1998. Callitrichaceae.429-431 [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Callitriche deflexa
BDJ.12.e115142-treatment1.xml (71.2KB, xml)
XML Treatment for Callitriche terrestris
BDJ.12.e115142-treatment2.xml (109.4KB, xml)
Supplementary material 1

Species occurrences

Hiroyuki Koga, Yuki Doll, Wataru Ohnishi, Horokazu Tsukaya

Data type

Table

Brief description

A list of examined herbarium samples.

File: oo_930177.xlsx

bdj-12-e115142-s001.xlsx (13.1KB, xlsx)
Supplementary material 2

Accession numbers of sequences used in phylogenetic analysis

Hiroyuki Koga, Yuki Doll, Wataru Ohnishi, Horokazu Tsukaya

Data type

Table

Brief description

Sequence information of the phylogenetic analysis.

File: oo_930178.xlsx

bdj-12-e115142-s002.xlsx (13.6KB, xlsx)

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