Journal of Plant Research

, Volume 130, Issue 1, pp 107–116 | Cite as

Molecular phylogeny of the cosmopolitan aquatic plant genus Limosella (Scrophulariaceae) with a particular focus on the origin of the Australasian L. curdieana

  • Yu Ito
  • Norio Tanaka
  • Dirk C. Albach
  • Anders S. Barfod
  • Bengt Oxelman
  • A. Muthama Muasya
Regular Paper


Limosella is a small aquatic genus of Scrophulariaceae of twelve species, of which one is distributed in northern circumpolar regions, two in southern circumpolar regions, two in the Americas, one endemic to Australia, and six in tropical or southern Africa or both. The Australasian L. curdieana has always been considered distinct but its close phylogenetic relationships have never been inferred. Here, we investigated the following alternative phylogenetic hypotheses based on comparative leaf morphology and habitat preferences or floral morphology: (1) L. curdieana is sister to the African L. grandiflora; or (2) it is closely related to a group of other African species and the northern circumpolar L. aquatica. We tested these hypotheses in a phylogenetic framework using DNA sequence data from four plastid DNA regions and the nuclear ITS region. These were analyzed using maximum parsimony and Bayesian inference. We obtained moderately resolved, partially conflicting phylogenies, supporting that accessions of L. grandiflora form the sister group to the rest of the genus and that L. curdieana groups with the African taxa, L. africana and L. major, and L. aquatica. Thus, the molecular evidence supports the second hypothesis. A biogeographic analysis suggests an out-of-southern Africa scenario and several dispersal events in the Southern Hemisphere. Past dispersal from southern Africa to Australasia is suggested, yet it cannot be excluded that a route via tropical Africa and temperate Asia has existed.


Aquatic plants Biogeography Dispersal Lamiales Phylogenetic inference 


Limosella L. (mudworts) is a genus in Scrophulariaceae with twelve species (Cook 2004; Glück 1934) that are either aquatic or amphibious in wetlands. The genus is distributed in temperate and subtropical regions. Their habitat and small seeds may have facilitated the global distribution of the taxon by migrating birds (Darwin 1872). The worldwide but discontinuous distribution includes one species in northern circumpolar regions, two in southern circumpolar regions, two in Americas, one endemic to Australia, and six in tropical or southern Africa or both (Cook 2004; Glück 1934; Fig. 1). Given the species richness of the genus and the distribution patterns of the related genera, such as Jamesbrittenia Kuntze, Lyperia Benth., Manulea L., Selago L. (Kornhall and Bremer 2004; Oxelman et al. 2005), it is reasonable to postulate that Limosella originated in southern Africa (Kornhall and Bremer 2004).
Fig. 1

Map of sampling localities of Limosella species. The main distribution areas of Limosella are roughly shaded with circles and letters referring to those used in Fig. 5. Species not included in this study are shown by area in grey font

While many of the species are either distributed in Africa or geographically close to Africa (Limosella aquatica L.), a few species are distributed in distant regions, i.e., L. americana Glück, L. curdieana F. Muell., and L. subulata E. Ives. Among these, L. americana described by Glück (1934) is sometimes recognized as a distinct taxon in Central and South America but was recently merged into L. aquatica (Brako and Zarucchi 1993; Cook 2004). Limosella subulata from North and South America has been recognized in some regional Floras (Brako and Zarucchi 1993; Crow and Hellquist 2000), but occasionally treated as a synonym of L. australis R.Br. (Cook 2004). Limosella curdieana, an Australasian endemic species, in contrast, is remarkable as it has never been synonymized, nor has its phylogenetic origin been inferred (Barker 1986, 1999; Harden 1992; Moore 1961).

Glück (1934) proposed an infrageneric classification of Limosella based on leaf morphology and habitat types, in which L. curdieana and L. grandiflora Benth (including its synonym, L. capensis Thunb.: Hilliard and Burtt 1986) are grouped together. On the basis of floral characters, however, L. curdieana appears to have affinities to the tropical African species, L. africana Glück and L. major Diels, as well as the circumpolar L. aquatica by having “petal lobes equal to or shorter than the sepals” (Figs. 2, 3; Barker 1986, 1999; Cook 2004; Godfrey and Wooten 1981; Gorshkova 1997; Harden 1992; Hong et al. 1998; Ivanina 2001; Moore 1961; Webb 1972; Yamazaki 1993).
Fig. 2

Comparison of key morphological features of the petiolate-leaved Limosella species. Usual morphological variation is indicated with boxes; maximum and minimum values extracted from published taxon descriptions are indicated with bars. a Lengths of petioles; b length of sepals; c length of petals; d length of capsules; e the ratio of petal length to sepal length; f the ratio of capsule length to sepal length. A vertical line show traits (mm) in ad and ratios in e, f. A: L. africana (Cook 2004); B: L. africana (Ghazanfar et al. 2008); C: L. major (Cook 2004); D: L. major (Philcox 1990); E: L. major (Ghazanfar et al. 2008); F: L. grandiflora (Cook 2004); G: L. infata (Cook 2004); H: L. vesiculosa (Cook 2004); I: L. aquatica (Hong et al. 1998); J: L. aquatica (Yamazaki 1993); K: L. aquatica (Gorshkova 1997); L: L. curdieana (Barker 1986, 1999; Harden 1992)

Fig. 3

Limosella curdieana in its natural habitat. Photos courtesy of the South Australian Seed Conservation Centre, Australia (

We aim to test these two competing hypotheses for Limosella curdieana in Australasia and examine whether the species is phylogenetically related to an African species, L. grandiflora, or to a Northern circumpolar species and other African species, i.e., L. africana and L. major. For that purpose, we employed simultaneous molecular phylogenetic analyses of plastid DNA (subsequently referred to as ptDNA) and nuclear ITS (subsequently referred to as nrITS) DNA sequences based on our worldwide taxon sampling of Limosella.

Materials and methods

Taxon sampling

Samples of Limosella were collected in the field or obtained from herbaria (Table 1). We follow a broadened taxonomic concept of twelve species in the genus. Because the only comprehensive revision made by Glück (1934) does not provide a key to the species, regional treatments were used for African (Cook 2004), Australasian (Barker 1986, 1999; Cook 2004; Harden 1992; Moore 1961), European (Webb 1972), North American (Crow and Hellquist 2000), and South American species (Cook 2004; Crow and Hellquist 2000). Yamazaki (1993) was consulted to confirm if our collection from Japan corresponded to the only species in the flora. Except for L. grandiflora with characteristic elongated stems (Cook 2004), positive identification of samples without flowers was problematic and these samples were treated as Limosella sp. Our taxon sampling, including four samples used in previous molecular phylogenetic studies (Kornhall and Bremer 2004; Oxelman et al. 2005), covers eight species: L. africana (3 specimens); L. aquatica (5; two from Europe, two from North America, and one from temperate Asia); L. australis (3; two from the Falklands and one from New Zealand); L. curdieana (2); L. grandiflora (4); L. macrantha R.E. Fr.(1); L. major (2); L. subulata (1); and Limosella sp. (1). The specimen UPS:BOT:V-120091, initially identified as L. grandiflora, was re-identified to be L. africana. In the present study we did not include specimens corresponding to L. americana in Central and South America, L. inflata Hilliard & B.L. Burtt and L. vesiculosa Hilliard & B.L. Burtt, both confined to small regions of South Africa, and L. longiflora, a widespread, close relative of L. australis (Cook 2004). Outgroup taxa were chosen following Kornhall and Bremer (2004) and Oxelman et al. (2005): species of Lyperia and Jamesbrittenia from the tribe Limoselleae Dumort., representing the sister genus to Limosella (Lyperia) and the basal genus of the tribe (Jamesbrittenia) for initial molecular phylogenetic analysis; those of Barthlottia, Chenopodiopsis, Cromidon, Dischisma, Glumicalyx, Hebenstretia, Jamesbrittenia, Lyperia, Manulea, Melanospermum, Microdon, Phyllopodium, Polycarena, Pseudoselago, Reyemia, Selago, Sutera, Tetraselago, Trieenea, and Zaluzianskya for species tree analysis (Table 1; Online resource 1). Glekia is not included due to the lack of nrITS data.
Table 1

Specimen and voucher information for the taxa included in this study










Jamesbrittenia megadenia


South Africa







Jamesbrittenia foliolosa


South Africa

Goldblatt P. & Porter L. 12488 (NBG)






Lyperia antirrhinoides


South Africa

Hagstroem & Acock 1162 (S)






Lyperia tristis


South Africa

Manning J.C. 2854 (NBG)






Limosella africana


South Africa: Eastern Cape; Elandsberg




LC133001 a



Limosella africana


Namibia: Hunsberge; Nuobrivier

W. Giess & M. Muller 14313 (PRE)






Limosella africana


South Africa: Central Cape; De Kom/Aarfontein

N.H. Helme 1541 (NBG)






Limosella aquatica


Hungary: South

YI1617 (TNS)






Limosella aquatica


Sweden: Östergötland







Limosella aquatica


Japan: Saitama; Koshigaya

TD4036 (TNS)






Limosella aquatica


USA: Nebraska







Limosella aquatica


USA: Nebraska







Limosella australis


UK: Falklands







Limosella australis


UK: Falklands







Limosella australis


New Zealand: Canterbury; Lake Forsyth

YI1769 (TNS)






Limosella curdieana


Australia: Victoria; Gunbower Isl.







Limosella curdieana


Australia: Queensland; South Glen







Limosella grandiflora


South Africa: Northeast Cape; Springbok

A. Le Roux 2355 (PRE)






Limosella grandiflora


South Africa: Western Cape; Riebeeckasteel







Limosella grandiflora


South Africa: Western Cape; Knolfontein







Limosella grandiflora


South Africa: Western Cape; Swartruggens







Limosella macrantha


Ethiopia: Bale Prov.; Bale Mts. Natl. park







Limosella major


Ethiopia: Begemdir Prov.; Simien







Limosella major


Ethiopia: Arsi-Robe/Sertu







Limosella subulata


Ecuador: Napo; Río Chalupas

Lagaard 101769 (AAU)






Limosella sp.


South Africa: Western Cape; Swartruggens

YI1991 (TNS)






Sequences generated in the present study are underlined. Herbarium acronyms are in accordance with Index Herbariorum (

aThis replaces AJ609170

DNA extraction, amplification, and sequencing

Total genomic DNA was extracted from silica gel-dried leaf tissues or from herbarium specimens using the CTAB method described in Ito et al. (2010). Parts of the plastid DNA regions, ndhF, rbcL, rps16 and trnT-trnF, and nrITS were PCR amplified with the following primers: ndhF-F2 (Oxelman et al. 1999) and (5′-CGATTATAKGACCAATTATATA) modified from Olmstead and Sweere’s (1994) ndhF-1955R for ndhF; rbcL-F1F (Wolf et al. 1994) and rbcL-1379R (Little and Barrington 2003) for rbcL; rps16-1F and rps16-2R (Oxelman et al. 1997); “a” and “b” for trnT-trnL and “c” and “f” for trnL-trnF (Taberlet et al. 1991); ITS-4 and ITS-5 for nrITS (Baldwin 1992). PCR amplification was performed using TaKaRa Ex Taq polymerase (TaKaRa Bio, Shiga, Japan), and PCR cycling conditions were 94 °C for 60 s; then 30 cycles of 94 °C for 45 s, 52 °C for 30 s, 72 °C for 60 s; and finally 72 °C for 5 min. The PCR products were cleaned using ExoSAP-IT (GE Healthcare, Piscataway, NJ, USA) purification and amplified using ABI PRISM Big Dye Terminator ver. 3.1 (Applied Biosystems, Foster City, CA, USA) with the same primers as those used for the PCR amplifications. DNA sequencing was performed with an ABI PRISM 377 DNA sequencer (Applied Biosystems). Automatic base-calling was checked by eye using Genetyx-Win ver. 3 (Software Development Co., Tokyo, Japan). The sequences generated and their metadata were submitted to the DNA Data Bank of Japan (DDBJ), which is a GenBank data provider (Table 1).

Data analysis

We assembled two datasets of Limosella: (1) ptDNA (ndhF, rbcL, rps16, and trnT-trnF); and (2) nrITS. Sequences were aligned using Mafft ver. 7.058 (Katoh and Standley 2013) and then inspected manually. We used “leave gappy regions” option in Mafft to code gaps found in ndhF, rps16, trnT-trnF, and nrITS.

The incongruence length difference test (Farris et al. 1994) in PAUP* ver. 4.0b10 (Swofford 2002) was employed to test for phylogenetic congruence among the four ptDNA regions using a partition homogeneity test with 1,000 replicates. This test did not reveal significant heterogeneity among genes (P value >0.05), and all subsequent analyses were therefore performed with a combined data set of ptDNA.

Phylogenetic inference was performed using maximum parsimony (MP) in PAUP* (Swofford 2002) and Bayesian inference (BI; Yang and Rannala 1997). In the MP analysis, a heuristic search was performed with 100 random addition replicates involving tree-bisection-reconnection (TBR) branch swapping, with the MulTrees option in effect. The MaxTrees option was set at 100,000. Bootstrap analyses (Felsenstein 1985) were performed using 1,000 replicates with TBR branch swapping and simple addition of sequences. The MaxTrees option was set to 1,000. Gaps were treated as binary characters.

BI analyses were conducted with MrBayes ver. 3.2.2 (Ronquist and Huelsenbeck 2003; Ronquist et al. 2012) run on the CIPRES portal (Miller et al. 2010) after the best models had been determined in MrModeltest ver. 3.7 (Nylander 2002); these models were GTR + I + Γ and GTR + Γ for ptDNA and nrITS, respectively. For gap characters, the “datatype = standard” option of MrBayes was used and default prior settings were applied, i.e., “ratepr = variable”. Analyses were run for 670,000 and 415,000 generations for ptDNA and nrITS, respectively, until the average standard deviation of split frequencies dropped below 0.01, sampling every 1,000 generations and discarding the first 25% as burn-in. The convergence and effective sampling sizes (ESS) of all parameters were checked in Tracer ver. 1.6 (Rambaut et al. 2014). All trees were visualized using FigTree ver. 1.3.1 (Rambaut 2009). Nodes are recognized as strongly, moderately, or weakly supported with: ≥95% bootstrap support (BS), ≥0.99 Bayesian posterior probabilities (PP); ≥70% BS, ≥ 0.95 PP; or <70% BS; <0.95 PP, respectively. The data matrices and the MP and BI trees are available at Treebase (TB2:S19401).

Species trees considering all samples were reconstructed for biogeographic analysis. Limosella macrantha was excluded due to incongruent positions between ptDNA and nrITS trees (see “Results”). A multispecies coalescent method (Heled and Drummond 2010) implemented in BEAST ver. 1.7.2 (Drummond et al. 2006; Drummond and Rambaut 2007) was performed. We ran *BEAST using the two data sets (ptDNA and nrITS) from 21 samples from seven ingroup taxa and assigning them to six terminal species, namely L. aquatica, L. australis, L. curdieana, L. grandiflora, L. major, plus Limosella sp.

We performed two independent runs of ten million generations of the MCMC chains, sampling every 1,000 generations. Convergence of the stationary distribution was checked by visual inspection of plotted posterior estimates using Tracer ver. 1.5 (Rambaut and Drummond 2007). After discarding the first 1,000 trees as burn-in, the samples were summarized in the maximum clade credibility tree using TreeAnnotator ver. 1.6.1 (Drummond and Rambaut 2007) with a posterior probability limit of 0.5 and summarizing mean node heights. The results were visualized using FigTree ver. 1.3.1 (Rambaut 2009).

Biogeographic analysis

Reconstruction of historical biogeography of Limosella was performed using RASP ver. 3.2 (Reconstruct Ancestral State in Phylogenies) (Yu et al. 2015). The Bayesian Binary Method (BBM; Ronquist and Huelsenbeck 2003) was selected because it: (1) accepts polytomies; (2) tends to suggest single distribution areas for ancestral nodes more often than others (Müller et al. 2015); and (3) is capable of providing unambiguous and informative results (Ito et al. 2016). BBM was conducted using the post burn-in species trees that resulted from the *BEAST analysis. The following seven biogeographic areas were defined: (a) Europe; (b) temperate Asia; (c) North America; (d) tropical Africa; (e) southern Africa; (f) Australasia; (g) South America (including Falklands). For the distribution of the species we used the area including the locality of accessions from the molecular phylogenetic analysis of the present study. Multiple ancestral states were allowed. The number of generations was set to 10 million and the first 10% of the samples were discarded as burn-in. All other parameters were kept at default settings.


Molecular phylogeny

Sequences of the concatenated four ptDNA regions of Limosella consisting of 22 ingroup and two outgroup samples resulted in an alignment with a total length of 5,067 bp. In total 428 characters including 29 binary-coded indels were polymorphic, of which 214 were parsimony-informative. Percentage of missing characters and gaps were: 15.20 and 0.00% (rbcL); 22.37 and 9.21% (rps16); 19.64 and 12.74% (trnL); 46.58 and 0.30% (ndhF) (Table 2). Analysis of this data set yielded the imposed limit of 100,000 MP trees (tree length = 466 steps; consistency index = 0.96; retention index = 0.96). The strict-consensus MP tree and MrBayes BI 50% consensus trees showed no incongruent phylogenetic relationships. Therefore, the better resolved MrBayes tree is presented (Fig. 4a).
Table 2

Percentage of missing characters and gaps by DNA regions


Missing characters

Total charactersa

Percentage of missing characters (%)

Gap length

Total alignment length

Percentage of gaps (%)




































aGaps are excluded

Fig. 4

MrBayes trees of Limosella based on a plastid DNA and b nuclear ITS. Branch lengths are proportional to molecular divergence among accessions. Numbers above or below the branches indicate bootstrap support (BS) calculated in maximum parsimony and Bayesian posterior probabilities (PP). BS < 50% and PP < 0.9 are indicated by hyphens while those of ≥95% and ≥0.99 are asterisks. Moderately- to strongly-supported groups are surrounded by round rectangles in background in gray and numbered. It should be noted, however, that group III is paraphyletic in ptDNA due to the inconsistent position of L. macrantha (see “Discussion”)

Limosella was divided into two strongly-supported groups: a clade of four accessions of L. grandiflora (group I; 100% BS; 1.0 PP) and the rest of the genus (100% BS; 1.0 PP). In the latter clade, Limosella sp. was placed as sister to the remaining accessions (89% BS; 1.0 PP). The other supported groups were (1) L. australis and L. subulata (group II; 88% BS; 1.0 PP) and (2) L. aquatica, L. curdieana, L. macrantha, and L. major (group III plus L. macrantha; <50% BS; 0.98 PP). Three accessions of L. africana were resolved in a polytomy with L. australis-L. subulata and L. aquatica-L. curdieana-L. macrantha-L. major.

The nrITS alignment consisting of 22 ingroup and two outgroup sequences had a total length of 691 bp. In total 121 characters including four binary-coded indels were polymorphic, of which 63 were parsimony-informative. Percentage of missing characters and gaps were: 5.63 and 1.75%, respectively (Table 2). Analysis of this data set yielded 22 MP trees (tree length = 166 steps; consistency index = 0.86; retention index = 0.89). The strict-consensus MP tree and MrBayes BI 50% consensus trees showed no incongruent phylogenetic relationships. Therefore, the better resolved MrBayes tree is presented (Fig. 4b).

A topology similar to that of ptDNA was recovered, with two strongly-supported clades: one comprising four accessions of Limosella grandiflora (group I; 87% BS; 1.0 PP) and another consisting of the remaining taxa (99% BS; 1.0 PP). In the clade of 17 accessions, L. africana UPS:BOT:V-120091 was placed as sister to the remaining accessions (68% BS; 0.99 PP). A clade of L. macrantha and L. africana β (87% BS; 0.98 PP) positioned sister to the remaining accessions (<50% BS; 1.0 PP). Group II, consisting of L. australis and L. subulata (88% BS; 0.99 PP) and group III (L. aquatica, L. curdieana, and L. major; 83% BS; 1.0 PP) formed a clade in the BI analysis (<50% BS; 0.97 PP), which was placed as sister to L. africana N.H. Helme 1541 (NBG).

The *BEAST species tree analysis retrieved two lineages: (a) BCDGHMPRSTZ (Barthlottia; Chenopodiopsis; Cromidon; Dischisma; Glumicalyx; Hebenstretia; Manulea; Melanospermum; Microdon; Phyllopodium; Polycarena; Pseudoselago; Reyemia; Selago; Sutera; Tetraselago; Trieenea; Zaluzianskya); (b) Limosella and Lyperia (Fig. 5).
Fig. 5

Bayesian *BEAST species tree for Limosella based on plastid DNA and nuclear ITS data. The outgroup clade (BCDGHMPRSTZ: Barthlottia; Chenopodiopsis; Cromidon; Dischisma; Glumicalyx; Hebenstretia; Manulea; Melanospermum; Microdon; Phyllopodium; Polycarena; Pseudoselago; Reyemia; Selago; Sutera; Tetraselago; Trieenea; Zaluzianskya) has been collapsed. Values above or below the branches represent the Bayesian posterior probabilities (PP). PP ≥ 0.95 are indicated by asterisks. Clades without significant support (PP < 0.75) are collapsed. Ancestral areas inferred from Bayesian Binary MCMC (BBM) are shown on each node. Multiple posterior results are shown in a box. The area codes follow Fig. 1 (A Europe; B temperate Asia; C North America; D tropical Africa; E southern Africa; F Australasia; G Central and South America)

Biogeographic reconstruction

The ancestral area of Limosella was inferred in southern Africa (1.0 PP); ambiguous results were obtained for the ancestral area of the most recent common ancestor (MRCA) of L. aquatica, L. curdieana, and L. major with highest probability for Australia (70.9 PP) (Fig. 5; Online resource 2).


Phylogeny of Limosella and the position of L. curdieana

The present study reconstructed the most detailed molecular phylogeny of the genus Limosella to date with a primary aim to test two competing hypotheses for ancestral relationships of the Australasian species, L. curdieana, i.e., whether the species is phylogenetically closely related to (1) the African species, L. grandiflora, or (2) a group composed of the two African species, L. africana and L. major, and the Northern circumpolar species, L. aquatica. The topologies recovered based on ptDNA and nrITS, respectively, are mostly congruent, except for a single accession of L. macrantha (see below). Both topologies resolve L. grandiflora as sister to the remaining species, including L. africana, L. aquatica, L. curdieana, and L. major (Fig. 4). The molecular evidence thus clearly rejected the hypothesis proposed by Glück (1934) and instead supported a relationship of L. curdieana with L. africana, L. major and L. aquatica.

Topological incongruence between ptDNA and nrITS

We detected a topological incongruence caused by our single accession of Limosella macrantha. In the ptDNA phylogeny this tropical African species (Ghazanfar et al. 2008; Glück 1934) is resolved in a clade including multiple accessions of the likewise tropical African species, L. major, whereas in the ITS phylogeny it is sister to southern African L. africana. Although morphological evidence suggests a close relationship between L. macrantha and L. australis (including specimens previously segregated as L. subulata) (Cook 2004; Glück 1934), the single accession of L. macrantha obtained from previous phylogenetic studies (Kornhall and Bremer 2004; Oxelman et al. 2005) was resolved as distantly related to L. australis (Fig. 4). Further taxon and data sampling will reveal whether this single specimen is part of an introgressive hybrid swarm or represents a hybrid. Alternatively, incomplete lineage sorting cannot be excluded as cause of the topological conflicts since this is common in closely related lineages such as Limosella.

Biogeography of Limosella and implications for dispersal

The present study supports a basal diversification of Limosella in southern Africa with moderate to strong support (Fig. 5), confirming a southern African origin of the genus as indicated by species richness of the ingroup and the distribution of sister genera. Thus, southern Africa seems to be a cradle for not only a number of drought-adapted taxa such as Crassulaceae (Mort et al. 2001), Scrophulariaceae (Oxelman et al. 2005), Thesium L. (Moore et al. 2010), Amaryllidoideae (Rønsted et al. 2012), and Asparagus L. (Norup et al. 2015) but also aquatics such as Limosella.

Bell et al. (2010) provided an estimated origin of Scrophulariaceae earlier than the mid-Paleogene, since tribes Myoporeae and Scrophularieae diversified ca. 51–53 mya. As Limosella is derived much later in the evolution of the family (Oxelman et al. 2005), vicariance due to plate tectonics is not an option to explain the discontinuous distribution worldwide and instead seed dispersal is more likely as is mentioned by Cook (2004): “the disseminules are small seeds, dispersed in mud and perhaps otherwise.” The current distribution of L. australis (including L. subulata) in e.g., Ecuador, New Zealand, and the Falklands could be a case of “wind highways” existing in the Southern Hemisphere (Muńoz et al. 2004).

The biogeographic origin of Limosella curdieana remains uncertain, but a close relationship with L. aquatica from Europe, temperate Asia, and North America, and L. major from tropical Africa (Ethiopia) is moderately supported (Fig. 4). The biogeographic analysis suggests Australasia as the ancestral area for the MRCA of the three species and thus implies a dispersal route from southern Africa to Australasia, and then to temperate Asia and tropical Africa (Fig. 5). Alternatively, considering the facts that (1) migratory bird routes are well documented between Asia and Australasia (Boere and Stroud 2006), (2) case studies exist that reveal disjunct distributions between Asia and Australasia (Lobelia L.: Kokubugata et al. 2012; Solenogyne Cass.: Nakamura et al. 2012), and (3) young plant groups in Australia have predominantly migrated from Asia (Crisp and Cook 2013), dispersal to Australasia via temperate Asia may also explain the geographic isolation of L. curdieana in Australia.

Implications for taxonomy of Limosella

Our molecular phylogenetic results provide additional insights into the taxonomy of Limosella. The three specimens of L. africana show close relationships, especially in ptDNA analysis, but do not form a clade in either of the analyses. No significant morphological differences are observed among the specimens except leaf shape, i.e., linear-lanceolate leaves for L. africana α and ovate leaves for L. africana β and L. africana γ. Still, all three specimens could belong to L. africana var. africana Glück, even in the strict sense, because none of these have much-clustered, short-pedicellate flowers or fruits (Cook 2004; Glück 1934). The sterile Limosella sp. shows affinities to L. africana var. africana in vegetative morphology, i.e., ovate leaves. Additional taxon sampling in flower and fruit from the locality and/or nearby habitats will provide further insight into the affinities of this accession, e.g., whether it belongs to either of the varieties, or represents an undescribed taxon. Since non-monophyletic taxa may be the result of various processes such as incomplete lineage sorting, a proper investigation of the species status of L. africana would require sampling from multiple loci in a species tree context.

The single accession of L. subulata from Ecuador forms a clade with three accessions of L. australis from New Zealand and the Falklands. This result agrees with the taxonomic treatment by Cook (2004) who synonymized L. subulata with the widespread L. australis. Further studies based on increased taxon and data sampling focusing on the taxonomic status of these taxa are needed.


Within a phylogenetic framework we provided new insights into the evolutionary origin of L. curdieana, the only Australasian endemic species in the genus. Our molecular phylogenetic analyses based on ptDNA and nrITS data sets revealed a basal diversification in southern Africa and close relationships between L. curdieana and northern circumpolar as well as a species from tropical Africa. The biogeographic analysis points to an out-of-southern Africa into the Northern Hemisphere and subsequent dispersal back into the Southern Hemisphere. Past dispersal from southern Africa to Australasia is suggested, however, we cannot exclude the existence of a route via tropical Africa and Asia.



The authors thank R. Kaul (NEB) for providing Limosella materials, T. Trinder-Smith (BOL), J. Palmer (CANB), C. Gallagher, P. Milne (MEL), C. Cupido (NBG), and E. van Wyk (PRE) for arranging loans from their institutions and/or for hospitality during our research visits; M. Hjertson (UPS) for sending scanned images of voucher specimens of Limosella at UPS; C. Ishii (Tsukuba) for help with DNA sequencing; J. Guerin (South Australian Seed Conservation Center, Australia) for providing L. curdieana photos. We would also like to thank P. B. Pelser (CANU), J. Murata, H. Ikeda, and T. Ohi-Toma (TI), and J. Li (Kunming) for their continuous encouragements and supports. YI and AMM received plant collecting permit in Western Cape, South Africa from Cape-Nature. This research was supported by FY 2012 Researcher Exchange Program between Japan society for the promotion of science (JSPS) and Royal Society of New Zealand (RSNZ) to YI, JSPS KAKENHI Grant Number 25440224 to NT, and the South African National Research Foundation (NRF) to AMM.

Supplementary material

10265_2016_872_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 17 kb)
10265_2016_872_MOESM2_ESM.docx (251 kb)
Supplementary material 2 (DOCX 251 kb)


  1. Baldwin BG (1992) Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: an example from the Compositae. Mol Phylogenet Evol 1:3–16CrossRefPubMedGoogle Scholar
  2. Barker WR (1986) Limosella. In: Jessop JP, Toelken HR (eds) Flora of South Australia, vol 3., Polemoniaceae to compositeABRS/CISRO, Melbourne, pp 1282–1284Google Scholar
  3. Barker WR (1999) Limosella. In: Walsh NG, Entwisle TJ (eds) Flora of Victoria, vol 4., Dicotyledons cornaceae to asteraceaeInkata Press, Port Melbourne, pp 497–498Google Scholar
  4. Bell CD, Soltis DE, Soltis PS (2010) The age and diversification of the angiosperms re-revisited. Am J Bot 97:1296–1303CrossRefPubMedGoogle Scholar
  5. Boere GC, Stroud DA (2006) The flyway concept: what it is and what it isn’t. In: Boere GC, Galbraith CA, Stroud DA (eds) Waterbirds around the world. The Stationery Office, Edinburgh, pp 40–47Google Scholar
  6. Brako L, Zarucchi JL (1993) Catalogue of the flowering plants and gymnosperms of Peru. Monographs in Systematic Botany volume 45, Missouri Botanical Garden, St. Louis, MOGoogle Scholar
  7. Cook CDK (2004) Aquatic and wetland plants of southern africa. Backhuys Publishers, LeidenGoogle Scholar
  8. Crisp MD, Cook LG (2013) How was the Australian Flora assembled over the last 65 million years? A molecular phylogenetic perspective. Annu Rev Ecol Evol Syst 44:303–324CrossRefGoogle Scholar
  9. Crow GE, Hellquist CB (2000) Limosella. In: Crow GE, Hellquist CB (eds) Aquatic and wetland plants of northeastern North America, vol 1., Pteridophytes, gymnosperms, and angiosperms: dicotyledonsThe University of Wisconsin Press, Madison, pp 327–329Google Scholar
  10. Darwin C (1872) The origin of species by means of natural selection. John Murray, LondonGoogle Scholar
  11. Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7:214–222CrossRefPubMedPubMedCentralGoogle Scholar
  12. Drummond AJ, Ho SYW, Phillips MJ, Rambaut A (2006) Relaxed phylogenetics and dating with confidence. PLoS Biol 4:e88CrossRefPubMedPubMedCentralGoogle Scholar
  13. Farris JS, Källersjö M, Kluge AG, Bult C (1994) Testing significance of incongruence. Cladistics 10:315–319CrossRefGoogle Scholar
  14. Felsenstein J (1985) Confidence limits on phylogenies—an approach using the bootstrap. Evolution 39:783–791CrossRefGoogle Scholar
  15. Ghazanfar SA, Hepper FN, Philcox D (2008) Scrophulariaceae. In: Beentje HJ, Ghazanfar SA (eds) Flora of tropical East Africa. Published on behalf of the East African governments by Royal Botanic Gardens, Kew, UK, pp 1–211Google Scholar
  16. Glück K (1934) Novae species et varietates generis Limosellae. Notizblatt des Botanischen Gartens und Museums zu Berlin-Dahlem 12:71–78CrossRefGoogle Scholar
  17. Godfrey RK, Wooten JW (1981) Limosella. In: Godfrey RK, Wooten JW (eds) Aquatic and wetland plants of southeastern United States Dicotyledons. Univ Georgia Press, Athens, p 649Google Scholar
  18. Gorshkova SG (1997) Limosella L. In: Schischkin BK, Bobrow EG (eds) Flora of U.S.S.R. vol 22, pp 367–369Google Scholar
  19. Harden GJ (1992) Limosella. Flora of New South Wales, vol 3. New South Wales University Press, Australia, pp 563–564Google Scholar
  20. Heled J, Drummond A (2010) Bayesian inference of species trees from multilocus data. Mol Biol Evol 27:570–580CrossRefPubMedGoogle Scholar
  21. Hilliard OM, Burtt BL (1986) Notes on some plants of southern Africa chiefly from Natal: XII. Notes R Bot Gard Edinb 43:189–228Google Scholar
  22. Hong DY, Yang H, Jin CL, Holmgren NH (1998) Scrophulariaceae. In: Wu ZY, Raven PH (eds) Flora of China. Science Press, Beijing, pp 1–212Google Scholar
  23. Ito Y, Ohi-Toma T, Murata J, Tanaka N (2010) Hybridization and polyploidy of an aquatic plant, Ruppia (Ruppiaceae), inferred from plastid and nuclear DNA phylogenies. Am J Bot 97:1156–1167PubMedGoogle Scholar
  24. Ito Y, Tanaka N, García-Murillo P, Muasya AM (2016) A new delimitation of the Afro-Eurasian plant genus Althenia to include its Australasian relative, Lepilaena (Potamogetonaceae)—Evidence from DNA and morphological data. Mol Phylogenet Evol 98:261–270CrossRefPubMedGoogle Scholar
  25. Ivanina LI (2001) Limosella. In: Fedorov AA (ed) Flora of Russia, the European part and bordering regions, vol 5. A. A. Balkema, Rotterdam, pp 325–347Google Scholar
  26. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kokubugata G, Nakamura K, Forster PI, Hirayama Y, Yokota M (2012) Antitropical distribution of Lobelia species (Campanulaceae) between the Ryukyu Archipelago of Japan and Oceania as indicated by molecular data. Aust J Bot 60:417–428CrossRefGoogle Scholar
  28. Kornhall P, Bremer B (2004) New circumscription of the tribe Limoselleae (Scrophulariaceae) that includes the taxa of the tribe Manuleeae. Bot J Linn Soc 146:453–467CrossRefGoogle Scholar
  29. Little DP, Barrington DS (2003) Major evolutionary events in the origin and diversification of the fern genus Polystichum (Dryopteridaceae). Am J Bot 90:508–514CrossRefPubMedGoogle Scholar
  30. Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES science gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, USA, pp 1–8Google Scholar
  31. Moore LB (1961) Limosella. In: Allan HH (ed) Flora of New Zealand, vol l., Government printerWellington, New Zealand, pp 846–847Google Scholar
  32. Moore TE, Verboom GA, Forest F (2010) Phylogenetics and biogeography of the parasitic genus Thesium L. (Santalaceae), with an emphasis on the Cape of South Africa. Bot J Linn Soc 162:435–452CrossRefGoogle Scholar
  33. Mort ME, Soltis DE, Soltis PS, Francisco-Ortega J, Santos-Guerra A (2001) Phylogenetic relationships and evolution of Crassulaceae inferred from matK sequence data. Am J Bot 88:76–91CrossRefPubMedGoogle Scholar
  34. Müller S, Salomo K, Salazar J, Naumann J, Jaramillo MA, Neinhuis C, Feild TS, Wanke S (2015) Intercontinental long-distance dispersal of Canellaceae from the New to the Old World revealed by a nuclear single copy gene and chloroplast loci. Mol Phylogenet Evol 84:205–219CrossRefPubMedGoogle Scholar
  35. Muńoz J, Felicisimo ÁM, Cabezas F, Burgaz AR, Martínez I (2004) Wind as a long- distance dispersal vehicle in the Southern Hemisphere. Science 304:1144–1147CrossRefPubMedGoogle Scholar
  36. Nakamura K, Denda T, Kokubugata G, Forster PI, Wilson GW, Peng C, Yokota M (2012) Molecular phylogeography reveals an antitropical distribution and local diversification of Solenogyne (Asteraceae) in the Ryukyu Archipelago of Japan and Australia. Biol J Linn Soc 105:197–217CrossRefGoogle Scholar
  37. Norup MF, Petersen G, Burrows S, Bouchenak-Khelladi Y, Leebens-Mack J, Pires JC, Linder HP, Seberg O (2015) Evolution of Asparagus L. (Asparagaceae): out-of-South-Africa and multiple origins of sexual dimorphism. Mol Phylogenet Evol 92:25–44CrossRefPubMedGoogle Scholar
  38. Nylander JAA (2002) MrModeltest v.1.0. Program distributed by the author. Department of Systematic Zoology, Uppsala University, Uppsala. Available at:
  39. Olmstead RG, Sweere JA (1994) Combining data in phylogenetic systematics: an empirical approach using three molecular data sets in the Solanaceae. Syst Biol 43:467–481CrossRefGoogle Scholar
  40. Oxelman B, Lidén M, Berglund D (1997) Chloroplast rps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Plant Syst Evol 206:393–410CrossRefGoogle Scholar
  41. Oxelman B, Backlund M, Bremer B (1999) Relationships of the Buddlejaceae s. l. inferred from chloroplast rbcL and ndhF sequences. Syst Bot 24:164–182CrossRefGoogle Scholar
  42. Oxelman B, Komhall P, Olmstead RG, Bremer B (2005) Further disintegration of Scrophulariaceae. Taxon 54:411–425CrossRefGoogle Scholar
  43. Philcox D (1990) Limosella. In: Launert E, Pope GV (eds) Flora Zambesiaca, volume 8, part 2. Flora Zambesiaca Managing Committee, London, pp 73–75Google Scholar
  44. Rambaut A (2009) FigTree v1.3.1: Tree Figure Drawing Tool.
  45. Rambaut A, Suchard MA, Xie W, Drummond AJ (2014) Tracer. Ver 1.6.
  46. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574CrossRefPubMedGoogle Scholar
  47. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61:539–542CrossRefPubMedPubMedCentralGoogle Scholar
  48. Rønsted N, Symonds MRE, Birkholm T, Christensen SB, Meerow AW, Molander M, Mølgaard P, Petersen G, Rasmussen N, van Staden J, Stafford GI, Jäger AK (2012) Can phylogeny predict chemical diversity and potential medicinal activity of plants? A case study of Amaryllidaceae. BMC Evol 12:182CrossRefGoogle Scholar
  49. Swofford DL (2002) PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4.0b. Sinauer, Sunderland, Massachusetts, USAGoogle Scholar
  50. Taberlet P, Ludovic G, Pautou G, Bouvet J (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol Biol 17:1105–1109CrossRefPubMedGoogle Scholar
  51. Webb DA (1972) Limosella. In: Tutin TG, Burges NA, Chater AO, Edmondson JR, Heywood VH, Moore DM, Valentine DH, Walters SM, Webb DA (eds) Flora Europaea, vol 3, pp 205–216Google Scholar
  52. Wolf PG, Soltis PS, Soltis DE (1994) Phylogenetic relationships of Dennstaedtioid ferns: evidence from rbcL sequences. Mol Phylogenet Evol 3:383–392CrossRefPubMedGoogle Scholar
  53. Yamazaki T (1993) Limosella. In: Iwatsuki K, Yamazaki T, Boufford DE, Ohba H (eds) Flora of Japan, vol IIIa. Angiospermae Dicotyledoneae Sympetalae, Kodansha, p 334Google Scholar
  54. Yang Z, Rannala B (1997) Bayesian phylogenetic inference using DNA sequences: a Markov Chain Monte Carlo method. Mol Biol Evol 14:717–724CrossRefPubMedGoogle Scholar
  55. Yu Y, Harris AJ, Blair C, He X (2015) RASP (Reconstruct Ancestral State in Phylogenies): a tool for historical biogeography. Mol Phylogenet Evol 87:46–49CrossRefPubMedGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan 2016

Authors and Affiliations

  • Yu Ito
    • 1
    • 2
  • Norio Tanaka
    • 3
  • Dirk C. Albach
    • 4
  • Anders S. Barfod
    • 5
  • Bengt Oxelman
    • 6
  • A. Muthama Muasya
    • 7
  1. 1.Biological SciencesUniversity of CanterburyChristchurchNew Zealand
  2. 2.Xishuangbanna Tropical Botanical GardenThe Chinese Academy of SciencesKunmingPeople’s Republic of China
  3. 3.Tsukuba Botanical GardenNational Museum of Nature and ScienceTokyoJapan
  4. 4.Institute of Biology and Environmental Sciences (IBU)Carl von Ossietzky-University OldenburgOldenburgGermany
  5. 5.Department of BioscienceAarhus UniversityAarhus CDenmark
  6. 6.Department of Biological and Environmental SciencesUniversity of GothenburgGothenburgSweden
  7. 7.Department of Biological SciencesUniversity of Cape TownCape TownSouth Africa

Personalised recommendations