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Introduction

Elatine L. is one of the two genera in the Elatinaceae, a family in Malpighiales (Tucker, 1986; Davis & Chase, 2004), and contains ca. 15–25 ephemeral amphibious species (Heywood et al., 2007). To the present knowledge, ten native taxa occur in Europe; however, Flora Europea lists seven (Cook, 1968) and Euro+Med Plantbase nine native species (Uotila, 2009b). One taxon belongs to the subgenus Potamopithys (Adanson) Seub(E. alsinastrum L.), and the other taxa are classified into subgenus Elatine Seub. (=Hydropiper Moesz): E. triandra Schkuhr (sect. Triandra Seub. (=Crypta (Nutt.) Seub.); E. brochonii Clavaud, E. campylosperma Seub., E. gussonei (Sommier) Brullo, Lanfr., Pavone & Ronsisv., E. hexandra (Lapierre) DC., E. hydropiper L., E. hungarica Moesz, E. macropoda Guss., and E. orthosperma Düben (section: Elatinella Seub.). Two more taxa of sect. Elatinella occur in the New World (in North America E. californica A. Gray and in South American E. ecuadoriensis Molau). Other taxa classified to sect. Triandra mainly occur in temperate regions of the Old and New World, with the probable center of diversity in North and South America. E. ambigua is another taxon from Europe (Uotila, 2009b). It shows no substantial genetic differences in relation to E. triandra (Sramkó et al., 2016) an Asian species also occurring in Europe.

Elatine alsinastrum is characterized by whorled leaves; all other species have opposite leaves, and are mainly distinguished by number of stamens (three, six or eight) and number of perianth lobes (three or four). The shape of leaves is variable, oblong or roundish, petiolate or almost sessile, and depends on environmental conditions. Flowers are sessile or pedunculated, while tiny seeds are oblong, curved or horseshoe-shaped (Cook, 1968; Tucker, 1986).

Recently, Elatine species have been of interest to researchers because of their rarity throughout their range, relatively poorly known distribution and taxonomy, ecology, karyology and phylogenetic relationships (e.g.,  Popiela, 2005; Misfud, 2006; Uotila, 2009a; Uotila, 2010; Popiela & Łysko, 2010; Popiela & Łysko, 2011; Popiela et al., 2011; Popiela et al., 2012; Popiela, Łysko & Molnár, 2013; Popiela et al., 2015; Takács, 2013; Molnár et al., 2013; Molnár, Popiela & Lukács, 2013; Šumberova & Hrivnak, 2013; Kalinka et al., 2014; Kalinka et al., 2015; Cai et al., 2016; Sramkó et al., 2016). The above-mentioned authors emphasized that the erratic temporal appearance of Elatine species depends mainly on environmental factors; for example, plants develop as aquatic or terrestrial forms, and, moreover, they are morphologically variable depending on the phase of drying on the ground. This variability and the very small size of plants and short-lasting, tiny flowers often make proper identifications difficult. Earlier leaf length and shape, pedicel length and seed shape were widely used for identification of Elatine taxa (Seubert, 1845; Niedenzu, 1925). The importance of seed morphology in Elatine taxonomy has been emphasized by many authors: the degree of seed curvature (i.e., seed shape) and seed coat reticulation have been considered very importan tfor recognizing individual species (Cook, 1968; Uotila, 1974; Uotila, 2010; Tucker, 1986; Misfud, 2006; Molnár et al., 2013).

There have been only a few studies addressing morphological variability of Elatine taxa (Mason, 1956; Molnár et al., 2013; Molnár, Popiela & Lukács, 2013). Recently, (Molnár et al., 2015) examined the level of phenotypic plasticity in Elatine. Analysis of morphological differences between aquatic and terrestrial forms of individual species clearly showed that vegetative traits are highly influenced by environmental factors and only seed traits are stable within species. According to Molnár et al. (2015), only seed morphology (aside from generative characteristics) is valuable for taxonomic purposes.

Consequently, we studied seed morphometric characteristics of 10 Elatine species, including all native European taxa, as a part of comprehensive surveys on taxonomy and phytogeography of this genus that have been conducted by a Hungarian-Polish research team since 2010. We assumed that advanced and methodically uniform seed characteristics are taxonomically important in this genus. Our aims were to (i) find statistical differences between Elatine species relative to seed morphological features, (ii) evaluate intra- and interspeciesseed variability, and then (iii) construct a guide to identifying species based onseed morphological features. Due to the small size of seeds the study was made by using SEM micrographs.

Material & Methods

Plant material and cultivation

Plants studied were collected across Europe. In total, seeds were collected from all 10 Elatine species and from three populations each, with an exception of very rare E. brochonii and E. campylosperma, two populations, so altogether 28 populations were used for the study. The distance between the populations of each species ranged from approximately 10–2,000 km. For the localities of the original material, and the voucher specimens, see Table 1 and Fig. 1. The studied seeds were gathered directly from the field, or from cultivated plants grown from the original material; in some cases seeds from herbarium specimens were used. Culture was conducted at the Center for Molecular Biology at the University of Szczecin, Poland and/or a the Department of Botany at the University of Debrecen, Hungary. Plants were grown in climate-controlled culture chambers with 12 h/day light and 30,000 lux light intensity, temperatures: under light, 22 ± 2 °C, and under dark, 18 ± 2 °C.

Table 1:
The species of Elatine and their populations included in the study, with the acronyms of the populations used in text, figs and tables.
Nr Acronym Name Origin*,** Latitude Longitude Collector, voucher No. of seeds Approx. distance between two/three populations (km)
1. alsHU E. alsinastrum L. Hungary: Konyár** 47.31 21.67 Molnár V. A. DE- 22226 50 620
2. alsPL1 E. alsinastrum L. Poland: Staw Noakowski* 50.80 23.03 Popiela A. SZUB- 008756 50
3. alsPL2 E. alsinastrum L. Poland: Strzelczyn 53.01 14.54 Popiela A. SZUB- 015968 50
4. broMO E. brochonii Clavaud Morocco: Ben Slimane** 33.62 −7.07 Lukács B. A. DE-43230 44 420
5. broSP E. brochonii Clavaud Spain: San Silvestre de Guzmán** 37.4 −7.36 Molnár V. A. DE-37684 49
6. camIT E. campylosperma Seub. Italy: Sardegna, Gesturi** 39.73 9.03 Molnár V. A. DE-37423 47 1,380
7. camSP E. campylosperma Seub. Spain: El Rocio, Donana** 37.12 −6.49 Molnár V. A. DE-37681 50
8. gusMAL E. gussonei (Sommier) Brullo, Lanfr., Pavone & Ronsisv. Malta: Gózó: Ta’ Sannat** 36.01 14.25 Molnár V. A. & Lukács B. A. DE-43229 50 1,265
9. gusSP E. gussonei (Sommier) Brullo, Lanfr. Pavone & Ronsisv. Spain: Casar de Cáceres** 39.33 −6.25 Molnár V. A. DE-43231 50
10. gusIT E. gussonei Italy: Sicily, Modica** 36.76 14.77 Molnár V. A. DE-38750 50
11. hexPL1 E. hexandra (Lapierre)DC. Poland: Janików (Janikowo) 51.57 14.96 Popiela A. SZUB- 015964 33 115
12. hexPL2 E. hexandra (Lapierre) DC. Poland: Milicz* 51.55 17.35 Popiela A. SZUB- 010851 50
13. hexPL3 E. hexandra (Lapierre) DC. Poland: Ruda Milicka* 51.53 17.34 Dajdok Z. SZUB- 011097 50
14. hunRUS E. hungarica Moesz Russia: Volgograd** 49.76 45.7 Mesterházy A. DE-37484 42 1,375
15. hunSLO E. hungarica Moesz Slovakia: Okánikowo 47.78 17.88 Eliáš P. SZUB- 010523 24
16. hunHU E. hungarica Moesz Hungary: Konyár** 47.31 21.67 Molnár V. A. DE-22266 50
17. hydHU E. hydropiper L. Hungary: Tiszagyenda** 47.36 20.52 Molnár V. A. DE-22273 39 550
18. hydPL1 E. hydropiper L. Poland: Parowa 51.38 15.23 Popiela A. 45
19. hydPL2 E. hydropiper L. Poland: Kwecko Lake 54.02 16.69 Popiela A.,Prajs B. SZUB- 015705 43
20. macIT E. macropoda Guss. Italy: Sardegna: Olmedo** 40.63 8.41 Molnár V. A. DE-37424 50 900
21. macSP1 E. macropoda Guss. Spain: Casar de Cáceres** 39.19 −6.29 Molnár V. A. DE-37692 46
22. macSP2 E. macropoda Guss. Spain: Mallorca: Cap Blanc/SaTore* 39.38 2.77 Popiela A., SZUB”-- 015969 42
23. ortCZ E. orthosperma Düben Czech Republic: Klášter* 49.02 15.15 Šumberova K. 45 1,260
24. ortFI1 E. orthosperma Düben Finland: Kokemäki 61.23 22.23 Suominen J, H 439800 25
25. ortFI2 E. orthosperma Düben Finland: Oulu* 65.06 25.47 Mesterházy A. DE-43232 50
26. triHU E. triandra Schkuhr Hungary: Kisköre* 47.50 20.50 Molnár V. A. DE-22282 41 570
27. triPL1 E. triandra Schkuhr Poland: Janików 51.57 14.96 Popiela A., SZUB- 010520 47
28. triPL2 E. triandra Schkuhr Poland: Bobięcińskie Małe Lake 54.01 16.82 Dambska I., SZUB- 010862 50
DOI: 10.7717/peerj.3399/table-1

Notes:

cultivation in Poland
cultivation in Hungary
Distribution of Elatine populations studied.

Figure 1: Distribution of Elatine populations studied.

For acronyms, see Table 1.

Elatine hungarica, E. hydropiper and E. triandra are protected species in Hungary and were sampled with the permission of the Hortobágy National Park Directorate (Permission id.: 45-2/2000, 250-2/2001).

To determinate the variability and diagnostic features of seeds, 24–50 seeds obtained from several individuals from each population (Table 1) were used. A total of more than 1,500 scanning electron microscope (SEM) images of the seeds were obtained at ×200 magnification using an SEM (Zeiss Evo, Molecular Biology and Biotechnology Center, University of Szczecin, Szczecin, Poland); however, 1,260 images were used for the morphometric study, because all cracked seeds were excluded. In total, six parameters were measured (Fig. 2): (A) object surface area; (B) profile specific perimeter (object circuit); (C) object rectangle a (length); (D) object rectangle b (width); (E) the angle of curvature; (F) number of pits on the seed coat counted in the middle row. Moreover, membrane presence and pit shape were evaluated.

The method of measuring of seed.

Figure 2: The method of measuring of seed.

(A) surface; (B) profile; (C) rectangle a; (D) rectangle b; (E) the angle of curvature (γ = α + β); (F) number of pits in the middle row.

To examine micromorphology of seed coat, 48 pictures at ×500, ×2,000, ×4,000, and ×7,000 were taken (Zeiss Evo SEM; Laboratory of Confocal and Electron Microscopy, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland)

Data analysis

To distinguish the characteristics that have the greatest impact on population and species discrimination, multiple discriminant analysis was used. Wilks λ was used to measure the discriminatory power of the model (0—perfect discrimination; 1—no discrimination). Interpretation of discriminant functions was performed using canonical analysis.

For visualization of the relationship between species and populations, Mahalanobis distance-based unweighted pair-group method using arithmetic averages to construct (UPGMA) trees was applied. Canonical values were shown using categorized scatterplots. The most discriminative traits were also independently tested by the non-parametric Kruskal–Wallis tests. All calculations were made in Statistica v. 12.5 software.

Results

Variability of populations within species

Discrimination analysis showed that all six traits significantly differentiate the populations studied (λ = 0.001, p < 0.001). Of these, the greatest contributions were as follows: number of pits, rectangle a (length), and the angle of curvature (Table 2).

Based on a Kruskal–Wallis tests, we found no statistically significant differences (p = 0.05) between populations studied of each European Elatine species regarding the following traits: (A) surface (all species except E. campylosperma, E. hungarica, E. hydropiper), (B) profile (all species except E. campylosperma, E. hexandra, E. hungarica, E. hydropiper), (C) rectangle a (all species except E. brochonii), (D) rectangle b (all species), (E) the angle of curvature (all species), (F) number of pits (all species except E. gussonei, E. hungarica, E. triandra) (Table 3). Accordingly, the traits studied did not show statistically significant variation between populationsof the following species: E. alsinastrum, E. macropoda and E. orthosperma.

Table 2:
Discriminant analysis of the studied populations of Elatine.
N = 1,260 λ = .00016F(162, 7217) = 153.07p < 0.0001
λ (Wilks) Fragm. (Wilks) F (27.1227) p Toler. R2
The angle of curvature 0.000461 0.342896 87.0870 0.0001 0.751829 0.248171
Rectangle a 0.000492 0.320751 96.2370 0.0001 0.254119 0.745881
Number of pits 0.000839 0.188320 195.8703 0.0001 0.989553 0.010447
Surface 0.000298 0.530541 40.2124 0.0001 0.169730 0.830270
Rectangle b 0.000229 0.690923 20.3291 0.0001 0.262387 0.737613
Profile 0.000199 0.793047 11.8592 0.0001 0.632832 0.367169
DOI: 10.7717/peerj.3399/table-2

However, there were large ranges of variation for some traits, especially within the following populations: E. orthosperma from Finland, Fin1 (for acronyms see Table 1) (surface: SD 36131.9 and rectangle a: SD 78.9), E. hungarica from Slovakia (profile: SD 498.1), E. triandra from Poland, PL1 (rectangle b:SD 39.9 and the angle of curvature: SD 33.7), and E. hydropiper from Hungary (number of pits: SD 5.3) (Fig. 3, Table 4). Conversely, the smallest ranges of variation were observed in the following populations: E. triandra from Poland, PL2 (surface: SD 5897.8), E. triandra from Hungary (rectangle a: SD 15.3), E. brochonii from Spain (profile: SD 51.7 and rectangle b: SD 13.6), E. hydropiper from Hungary and E. macropoda from Spain, SP2 (angle of curvature, SD 10.5; SD 10.3, respectively), and E. brochonii from Morocco (number of pits: SD 0.9) (Fig. 3, Table 4).

Multidimensional scaling based on a correlation matrix of Mahalanobis distance of the six features studied revealed the greatest similarity between the three populations of the following species: E. alsinastrum, E. macropoda, E. hexandra (Fig. 4).

Variability between species

Discriminant analysis showed that all variables could discriminate species (λ < 0.01). The greatest impact was from the following features: number of pits, the angle of curvature and rectangle a (Table 5).

Table 3:
Significant differences between populations of Elatine. based on Kruskal–Wallis tests (p = 0.05).
For acronyms, see Table 1.
alsHU alsPL1 alsPL2 brochMO brochSP camIT camSP gusIT gusMAL gusSP hexPL1 hexPL2 hexPL3 hunHU hunRUS hunSLO hydHU hydPL1 hydPL2 macIT macSP1 macSP2 ortCZ ortFI1 ortFI2 triHU triPL1 triPL2
alsHU abcf abcf cdef abcdef cde cdef acde acef abce abce abcde abcde bcde cdef cdef cdef bcde bef abce ef f ef abcd abce abcdef
alsPL1 abcf abcdf cdef abcdef cde cdef acde ac abc abce abcde abce cde bcdef abcdef cdef ce e ce f f f abcd abc abcd
alsPL2 abcf abcdf cdef abcdef cde cdef acde ac abc abce abcde abce cde cdef cdef cdef ce e abce f f f abcd abcd abcd
brochMO abcf abcf abcf c abcdef cdef abdef abde abdef be f ef cdef cdef de abdef abdef abdef abdef abcde def abcf abcf abcf cf f ace
brochSP abcf abcdf abcdf c abdef abdef abcdef abcde abcdef abce bcef bcef def def abde abcdef abcdef abcdef abcdef abcdef abcdef abcdef abcf abcdef f ef ef
camIT cdef cdef cdef abcdef abdef ab cf cef cf acdef abcdef abcdef abdf abdf bf a abcdef bcdef abcdef cde cde cde abdef abdef abdef
camSP abcdef abcdef abcdef cdef abdef ab abcf abcef abcf bcdef cdef cdef f f abc abc c cef acdef cdef bcde cde bcde abde adef abdef
gusIT cde cde cde abdef abcdef cf abcf f ade abde abd abcf abcdf f f af ad cde abd acdef cdef acdef abcdef abcde abcde
gusMAL cdef cdef cdef abde abcde cef abcef f ad abde abdf abcf abcf c ef ef ef f d abdf cdef def def abcdef abcdef abcde
gusSP acde acde acde abdef abcdef cf abcf f f adef abdef abde abcd abcd ab f f af abde abcdef abde acdef acdef acdef abcde abcde abcdef
hexPL1 acef ac ac be abce acdef bcdef ade ad adef b b bcdef bcef de adef adef def cf f cf abcdf abc abcd
hexPL2 abce abc abc f bcef abcdef cdef abde abde abdef b cdef cdef cde abdef abdef adef ade a abcf f abcf bcf c abcd
hexPL3 abce abce abce ef bcef abcdef cdef abd abdf abde b cd bcef cd abdef abdef def a abcf f abcf abcdef abc abcd
hunHU abcde abcde abcde cdef def abdf f abcf abcf abcd bcdef cdef cd abf abdf bf bc abcef c bcdef bcdef bcdef ade ade abdef
hunRUS abcde abce abce cdef def abdf abcdf abcf abcd bcef cdef bcef abf abcdf abcdf abcf abc abcef abc abcef abce abce de de def
hunSLO bcde cde cde de abde bf f c ab de cde cd abf bf abf f c c bcdef cdef cdef abde abde abde
hydHU cdef bcdef cdef abdef abcdef abc f ef f adef abdef abdef abf abcdf bf ab abdef bcdef abdef acde cde acde abdef abdef abcdef
hydPL1 cdef abcdef cdef abdef abcdef abc f ef f adef abdef abdef abdf abcdf abf ab abcdef abcdef abdef acde acde acde abdef abdef abcdef
hydPL2 cdef cdef cdef abdef abcdef a c af ef af def adef def bf abcf f ab ab def cdef def cde cde cde abdef abdef abcdef
macIT bcde ce ce abdef abcdef abcdef cef ad f abde ade bc abc c abdef abcdef def bcf ef ef abcde abcd abcd
macSP1 bef e e abcde abcdef bcdef acdef cde d abcdef a a abcef abcef c bcdef abcdef cdef bf f f abcdef abcd abcd
macSP2 abce ce abce def abcdef abcdef cdef abd abdf abde c abc abdef abdef def bcf ef bcf abcde abcd abcdf
ortCZ ef f f abcf abcdef cde bcde acdef cdef acdef cf abcf abcf bcdef abcef bcdef acde acde cde bcf bf bcf abcdf abcdf abcdf
ortFI1 f f f abcf abcf cde cde cdef def acdef f f f bcdef abce cdef cde acde cde ef f ef abcd abcf abcdf
ortFI2 ef f f abcf abcdef cde bcde acdef def acdef cf abcf abcf bcdef abce cdef acde acde cde ef f bcf abcd abcdf abcdf
triHU abcd abcd abcd cf f abdef abde abcdef abcdef abcde abcdf bcf abcdef ade de abde abdef abdef abdef abcde abcdef abcde abcdf abcd abcd f
triPL1 abce abc abcd f ef abdef adef abcde abcdef abcde abc c abc ade de abde abdef abdef abdef abcd abcd abcd abcdf abcf abcdf
triPL2 abcdef abcd abcd ace ef abdef abdef abcde abcde abcdef abcd abcd abcd abdef def abde abcdef abcdef abcdef abcd abcd abcdf abcdf abcdf abcdf f
DOI: 10.7717/peerj.3399/table-3

Notes:

a

surface

b

profile

c

rectangle a

d

rectangle b

e

angle of curvature

f

number of pits

Boxplots of the most discriminative seed traits among 28 studied populations of Elatine.

Figure 3: Boxplots of the most discriminative seed traits among 28 studied populations of Elatine.

Notations: boxes indicate 25–75 percentiles, white point indicate medians, whiskers exclude outliers, black points indicate outliers. For acronyms, see Table 1. (A) surface; (B) profile; (C) rectangle a; (D) rectangle b; (E) angle; (F) pits.
Multidimensional scaling based on a correlation matrix of Mahalanobis distance for seed traits among 28 populations of Elatine.

Figure 4: Multidimensional scaling based on a correlation matrix of Mahalanobis distance for seed traits among 28 populations of Elatine.

For acronyms, see Table 1
Table 4:
SD values of seed traits studied of the populations of Elatine.
For acronyms, see Table 1.
Acronym Surface (µm2) Profile (µm) Rectangle_a (µm) Rectangle_b (µm) The angle of curvature (°) Numer of pits
alsHU 22108.8 152.4 55.0 26.1 12.5 2.1
alsPL1 12280.7 69.8 34.2 22.7 16.8 1.3
alsPL2 23478.5 139.6 51.6 28.0 11.7 1.5
brochMO 14826.5 86.4 37.3 21.0 14.2 0.9
brochSP 7424.4 51.7 22.7 13.6 14.2 1.3
camIT 23754.8 154.9 37.4 34.7 21.4 5.0
camSP 12567.2 106.3 19.7 23.1 16.1 3.6
gusIT 23082.4 237.0 44.7 27.4 17.7 3.4
gusMAL 18526.5 172.1 39.8 24.8 23.0 1.6
gusSP 17567.0 121.0 29.1 26.0 26.9 4.1
hexPL1 12783.1 194.6 41.8 27.8 26.1 1.6
hexPL2 9186.5 87.2 27.3 26.0 23.6 1.7
hexPL3 10467.0 110.1 39.3 32.9 10.3 1.4
hunHU 7222.9 59.2 24.9 19.4 17.4 4.0
hunRUS 8318.2 84.2 17.5 17.8 17.4 2.7
hunSLO 24936.3 498.1 40.1 30.3 15.9 1.9
hydHU 11886.9 101.5 18.3 19.2 10.5 5.3
hydPL1 22799.7 252.3 35.7 30.5 16.7 4.2
hydPL2 12877.0 221.3 41.1 22.2 20.6 4.3
macIT 14752.8 121.9 45.0 30.6 21.4 2.1
macSP1 21061.9 153.9 45.6 22.4 18.3 1.8
macSP2 16799.6 160.0 41.1 35.2 10.3 2.5
ortCZ 18174.3 271.2 51.0 34.8 25.7 3.8
ortFI1 36131.9 241.2 78.9 24.5 24.3 3.0
ortFI2 17093.8 177.3 45.3 30.7 25.4 3.0
triHU 6365.6 67.3 15.3 13.9 16.4 2.0
triPL1 11403.4 140.7 36.4 39.9 33.7 1.8
triPL2 5897.8 55.6 22.1 18.5 11.7 2.0
DOI: 10.7717/peerj.3399/table-4
Table 5:
Discriminant analysis of seed traits studied of the European Elatine species.
N = 1,260 λWilks: .00252 F (54,6352) = 262.68 p < 0.0001
λ (Wilks) Fragm. (Wilks) F (9.1245) p Toler. R2
The angle of curvature 0.005051 0.499212 138.7702 0.0001 0.632234 0.367766
Rectangle a 0.004048 0.622838 83.7685 0.0001 0.298593 0.701407
Number of pits 0.007668 0.328805 282.3820 0.0001 0.916368 0.083632
Surface 0.002735 0.921836 11.7295 0.0001 0.153021 0.846979
Profile 0.002759 0.913793 13.0504 0.0001 0.186761 0.813239
Rectangle b 0.002717 0.927840 10.7585 0.0001 0.509627 0.490373
DOI: 10.7717/peerj.3399/table-5

The Kruskal–Wallis tests showed, in many cases, lack of statistical significance between species relative to the studied seed traits (Table 6). Regarding the trait surface, only E. triandra seeds showed statistical significance compared with all species tested. Analysis of all characteristics showed the least amount of statistically significant differences between the following species pairs: E. alsinastrum and E. orthosperma, E. hexandra and E. macropoda, as well as E. gussonei and E. hydropiper (Table 6).

Table 6:
Significant differences of studied features between the European species of Elatine based on Kruskal–Wallis tests ( p = 0.05).
E. alsinastrum E. brochonii E. campylosperma E. gussonei E. hexandra E. hungarica E. hydropiper E. macropoda E. orthosperma E. triandra
E. alsinastrum abcdf abcdef acdef abcef abcde cdef abcde aef abcde
E. brochonii abcdf abcdef abcdef bcdef cdef abdef abcdef abcdef acef
E. campylosperma abcdef abcdef abcef acdef abdef abc cdef bcde abdef
E. gussonei acdef abcdef abcef abde abcdf ef abcde acdef abcde
E. hexandra abcef bcdef acdef abde bcdef abcdef ad abcf abcdf
E. hungarica abcde cdef abdef abcdf bcdef abcdef abcdef abcdef abde
E. hydropiper cdef abdef abc ef abcdef abcdef abcdef acde abcdef
E. macropoda abcde abcdef cdef abcde ad abcdef abcdef bcdef abcde
E. orthosperma aef abcdef bcde acdef abcf abcdef acde bcdef abcdf
E. triandra abcde acef abdef abcde abcdf abde abcdef abcde abcdf
DOI: 10.7717/peerj.3399/table-6

Notes:

a

surface

b

profile

c

rectangle a

d

rectangle b

e

angle of curvature

f

number of pits

There was a large range of variation for the taxa studied regarding the following traits: seed size (traits: surface, profile, and rectangle a), especially within E. hungarica (SD 27183.7, SD 285.9, and SD 62.0, respectively); the angle of curvature, E. gussonei (SD 44.7); and number of pits, E. campylosperma (SD 7.2). The smallest variation was present in E. triandra (surface SD 14587.3, profile: SD 171.8, rectangle a: SD 54.6) and E. brochonii (rectangle b SD 20.6, the angle of curvature SD 14.7, pits: SD 1.3). The characteristics associated with size (surface, profile, rectangle a, rectangle b) revealed that the following species had the smallest seeds: E. brochonii and E. triandra, while the largest seeds in the studied species belonged to E. gussonei and E. hydropiper (Fig. 5, Table 7).

Boxplots of the most discriminative seed traits among Elatine species studied.

Figure 5: Boxplots of the most discriminative seed traits among Elatine species studied.

Notations: boxes indicate 25–75 percentiles, white points indicate medians, whiskers exclude outliers, black points indicate outliers. For acronyms, see Table 1. (A) surface; (B) profile; (C) rectangle a; (D) rectangle b; (E) angle; (F) pits.
Morphological relationships of seeds among surveyed Elatine species displayed by Mahalanobis distance-based UPGMA cluster based on the following features: rectangle a, angle of curvature, and number of pits.

Figure 6: Morphological relationships of seeds among surveyed Elatine species displayed by Mahalanobis distance-based UPGMA cluster based on the following features: rectangle a, angle of curvature, and number of pits.

For acronyms, see Table 1.
Table 7:
SD values for seeds traits of the European Elatine species.
Surface (µm2) Profile (µm) Rectangle_a (µm) Rectangle_b (µm) Angle of curvature (°) Number of pits
E. alsinastrum 20213.4 142.3 50.1 27.8 16.7 1.8
E. brochonii 20881.3 133.8 58.9 20.6 14.7 1.3
E. campylosperma 30981.9 219.0 35.1 56.7 20.8 7.2
E. gussonei 26873.7 184.4 44.7 46.1 44.7 4.5
E. hexandra 11356.3 165.5 37.1 30.6 26.0 1.6
E. hungarica 27183.7 285.9 62.0 35.7 20.3 3.7
E. hydropiper 31653.4 250.3 33.6 41.5 17.3 6.6
E. macropoda 19610.4 148.5 50.3 30.6 20.9 2.5
E. orthosperma 22819.4 239.2 60.9 31.3 26.2 4.2
E. triandra 14587.3 171.8 54.6 32.2 27.5 2.7
DOI: 10.7717/peerj.3399/table-7

The classification matrix of the discriminant analysis showed that the level of classification varied from 86% (rectangle a, the angle of curvature, number of pits) to 84% (surface, profile, rectangle a, rectangle b, the angle of curvature, number of pits). The highest values of classification were found for E. alsinastrum, E, brochonii, E. hydropiper, and E. orthosperma (all greater than 90%). The lowest values were found for E. campylosperma (57%, 55%) (Table 8).

Table 8:
Classification matrix based on discriminant function analysis of seeds traits of Elatine species.
E. alsinastrum E. brochonii E. campylosperma E. gussonei E. hexandra E. hungarica E. hydropiperer E. macropoda E. orthosperma E. triandra Correct n
Correct classification (%) A 96 94 57 85 79 85 94 83 95 88 86
B 97 92 55 78 71 82 95 78 93 91 84
E. alsinastrum A 144 1 0 0 3 0 0 2 0 0 150
B 145 1 0 0 4 0 0 0 0 0 150
E.brochonii A 1 87 0 0 5 0 0 0 0 0 93
B 1 86 0 0 5 0 0 0 0 1 93
E. campylosperma A 0 0 55 0 0 17 25 0 0 0 97
B 0 0 53 0 0 16 28 0 0 0 97
E. gussonei A 0 0 1 126 0 1 4 16 0 0 148
B 0 0 1 115 0 3 6 23 0 0 148
E. hexandra A 1 1 0 0 105 0 0 26 0 0 133
B 1 2 0 0 95 0 0 35 0 0 133
E. hungarica A 0 0 1 16 0 99 0 0 0 0 116
B 0 0 1 20 0 95 0 0 0 0 116
E. hydropier A 0 0 6 2 0 0 119 0 0 0 127
B 0 0 5 1 0 0 121 0 0 0 127
E. macropoda A 5 0 0 0 17 0 0 115 0 1 138
B 5 0 0 1 24 0 0 107 0 1 138
E. orthosperma A 4 0 0 0 1 0 0 1 114 0 120
B 6 0 0 0 1 0 0 1 112 0 120
E. triandra A 0 1 0 0 15 1 0 0 0 121 138
B 0 1 0 0 9 1 0 2 0 125 138
Total classified A 155 90 63 144 146 118 148 160 114 122 1,260
B 158 90 60 137 138 115 155 168 112 127 1,260
(Correct n) − (Tot. class.) A −5 3 34 4 −13 −2 −21 −22 6 16
B −8 3 37 11 −5 1 −28 −30 8 11
DOI: 10.7717/peerj.3399/table-8

Notes:

A

rectangle a, angle of curvature, pits

B

surface, profile, rectangle a, rectangle b, angle of curvature, number of pits

UPGMA clusters of Mahalanobis distance based on rectangle a, the angle of curvature, and number of pits yielded two groups: species with straight or nearly straight seeds, and species with curved and U-shaped seeds (Fig. 6). The greatest similarity was found between seeds of E. hexandra and E. macropoda, and E. campylosperma and E. hydropiper. The spatial distribution of observed characteristics of the analyzed species is depicted as a categorized scatterplot based on canonical analysis values (Fig. 7).

Categorized scatterplot based on canonical analysis value for seeds of the European species of Elatine.

Figure 7: Categorized scatterplot based on canonical analysis value for seeds of the European species of Elatine.

For acronyms, see Table 1.

Discussion

Our study shows that in Elatine tested seed variability is mainly associated with size-connected traits, especially surface, profile, rectangle b, and, to a lesser extent, rectangle a. This allowed us to draw the conclusion that to distinguish seeds of these species the most useful traits are the angle of curvature and number of pits, and to a lesser extent rectangle a (length). These findings confirm previous knowledge about the usefulness of these features in Elatine taxonomy (Misfud, 2006; Uotila, 2009a; Uotila, 2010; Molnár et al., 2013; Molnár et al., 2015). Nevertheless, our study revealed that the range of variation of European Elatine morphological features is large, both between species and the populations of each species.

Regarding intraspecific variability, the traits studied were not statistically significantly different between studied populations of the following taxa: E. alsinastrum, E. macropoda, E. hexandra. Conversely, E. gussonei, E. campylosperma E. hungarica and E. hydropiper seeds showed statistically significant intrapopulation variability. The taxonomic status of the first three species is still being elucidated. Elatine gussonei, an enigmatic plant of the Mediterranean, was first described as a variety of E. hydropiper and was later classified as a separate species (Brullo et al., 1988; Misfud, 2006; Molnár et al., 2013; Molnár, Popiela & Lukács, 2013). Elatine campylosperma was described by Seubert (1845) from Sardinia, and later greatly neglected by most researchers by synonymizing this species under E. macropoda; at present, it is considered a separate species (Kalinka et al., 2015). Elatine hungarica was last collected in 1960 and rediscovered in Hungary in 1998 (Molnár et al., 1999); for years its taxonomic status was under discussion (Molnár et al., 2013).

Our present study showed that regarding shape statistically only E. alsinastrum and E. orthosperma seeds are nearly straight and seeds of all other species are curved to varying degrees; the range of variation in some species is large in this respect, especially in E. gussonei, E. triandra, and E. hexandra.

If distinction of species is only based on seeds, it would be easy to confuse the following species pairs: E. alsinastrum and E. orthosperma, E. hexandra and E. macropoda, E. campylosperma and E. hydropiper, andE. gussonei and E. hungarica, especially if only a few seeds are evaluated. Previously, Misfud (2006), who worked on Malta and Mallorca populations, pointed out the importance of distinction based on greater seed curvature in E. gussonei compared with E. macropoda; although this is true if averages are used, there is a substantial amount of overlap in curvature and this could lead to confusion if the curvature of only few seeds are analyzed. This was also confirmed by our results. Misfud (2006) also drew attention to the distinctive seed testa reticulation, and claimed that the wide hexagonal shape of pits in E. gussonei and smaller number of pits/row (15 ± 3) are very difficult to confuse with E. macropoda’s 21 ± 3 narrow pits/row. Our study yielded different results: seeds of E. macropoda populations had similar number of pits [(13–)19–23–(29)] compared to E. gussonei [(17–)23(–32)]. However, because Misfud (2006) did not precisely describe the method of counting pits (especially in which row pits were counted), it is difficult to compare our results. Molnár et al. (2013) pointed out that seeds of E. hungarica are much more curved than those of E. gussonei, and especially of E. macropoda and E. orthosperma, but somewhat less curved than those of E. hydropiper. Our current study revealed that the range of variation for the feature the angle of curvature of E. hungarica seeds is similar to that of E. gussonei, and more curved seeds are found in E. hydropiper and E. campylosperma. These results are basically consistent with observations of Molnár et al. (2013), especially considering that more varied material was used in the current study. Our research confirms observations of Misfud (2006) and Molnár et al. (2013) concerning the evident semilunar membrane on the concave side of seeds (Figs. 811). The membrane was present and clearly visible in all highly curved fresh seeds of the following species: E. gussonei, E. hydropiper, E. hungarica, and E. campylosperma. Regarding the seed testa, a very distinctive network-shape ornamentation pattern of E. triandra as visible (Figs. 9J9L, 11). Elatine campylosperma seeds showed the most distinctive reticulation, and were characterized by a large number of narrow, rectangular pits (Fig. 10), and round-shaped pits (Figs. 8G8I). Similarly, rectangular-shaped pits were found in seeds of the following species: E. alsinastrum, E. triandra, E. orthosperma, E. hydropiper, and E. macropoda; however, hexagonal pits were also present (Figs. 10 and 11). The pit shapes of E. gussonei, E. hungarica, E. brochonii, and E. hexandra are usually both hexagonal and rectangular, with a predominance of the former (Figs. 10 and 11). Similar observations for some of these species were made by Misfud (2006), Molnár et al. (2013), Molnár, Popiela & Lukács (2013). We believe that the shape of pits may be an additional feature that helps distinguish seeds of individual species (Figs. 8 and 10). However, we found no diversity in seed coat micromorphology within pits (e.g., pores, strophioles) that could have potential taxonomic importance. Seed coats within pits were smooth with the exception of irregular strips, and the porosity of the seed coat is visible only in the inner layer of cracked seeds (Figs. 8Q8R, 9I). Ornamentation pattern (pit shape) becomes distinct as the seeds dry up. The outer layer of the seed coat is very thin and easily destroyed ( Figs. 9A9B; 9D9F).

The diversity in seed coat micromorphology of Elatine alsinastrum (a–alsHu; b, c–alsPL1), E. brochonii (a, b–broMO; c–broSP), E. campylosperma (a, b–camIT; c–camSP), E. gussonei (a, b–gusMAL; c–gusSP), E. hexandra (a, b–hexPL1; c–hexPL2), E. hungarica (a, b–hunR; c–hunSL).

Figure 8: The diversity in seed coat micromorphology of Elatine alsinastrum (a–alsHu; b, c–alsPL1), E. brochonii (a, b–broMO; c–broSP), E. campylosperma (a, b–camIT; c–camSP), E. gussonei (a, b–gusMAL; c–gusSP), E. hexandra (a, b–hexPL1; c–hexPL2), E. hungarica (a, b–hunR; c–hunSL).

Scale bar = 10 µm. For acronyms, see Table 1. (A–C) E. alsinastrum; (D–F) E. brochonii; (G–I) E. campylosperma; (J–L) E. gussonei; (M–O) E. hexandra; (P–R) E. hungarica.
The diversity in seed coat micromorphology of Elatine hydropiper (a - hydHu, b, c - hydPL1); E. macropoda (a, b -macIT; c–macSP), E. orthosperma (a, b -ortCZ; c - ortFI1), E. triandra (a–triHU; b, c–triPL1).

Figure 9: The diversity in seed coat micromorphology of Elatine hydropiper (a - hydHu, b, c - hydPL1); E. macropoda (a, b -macIT; c–macSP), E. orthosperma (a, b -ortCZ; c - ortFI1), E. triandra (a–triHU; b, c–triPL1).

Scale bar = 10 µm. For acronyms, see Table 1. (A–B) E. hydropiper; (D–F) E. macropoda; (G–I) E. orthosperma; (J–L) E. triandra.
The diversity of seeds of Elatine campylosperma, E. gussonei, E. hydropiper, E. hungarica, E. macropoda.

Figure 10: The diversity of seeds of Elatine campylosperma, E. gussonei, E. hydropiper, E. hungarica, E. macropoda.

Scale bar =200 µm. (A–E) E. campylosperma; (F–J) E. gussonei; (K–O) E. hydropiper; (P–T) E. hungarica; (U–Y) E. macropoda.
The diversity of seeds of Elatine orthosperma, E. alsinastrum, E. brochonii, E. hexandra, E. triandra.

Figure 11: The diversity of seeds of Elatine orthosperma, E. alsinastrum, E. brochonii, E. hexandra, E. triandra.

Scale bar =200 µm. (A–E) E. orthosperma; (F–J) E. alsinastrum; (K–O) E. brochonii; (P–T) E. hexandra; (U–Y) E. triandra.

Our research allowed us to construct a guide that can be useful to identify the studied taxa based on seed traits. We believe that this guide is important for better recognition of these rare and endangered species, and can be useful for elucidating the history of range formation of these taxa in the Holocene and their origin. Elatine subfossil finds were discovered in late-glacial and pre-boreal sediments in the last few centuries (Latałowa, 1992; Brinkkemper et al., 2008; Kowalewski et al., 2013). The ecological amplitude of this species provides robust clues for environmental reconstruction, which must have been a temporarily flooded fresh water area. “...since this type of environment is strongly threatened on a worldwide scale, the presence of these species in the past may also provide interesting information for present nature development projects...” (Brinkkemper et al., 2008).

Identification guide and descriptions for European species of Elatine based on seed morphology presented in Figs. 10 and 11. Note: the guide does not include exceptional values given in parentheses in the descriptions (min. outliers 1.5) 25%–75% (max. outliers 1.5).

1 Seeds straight –almost straight –slightly curved, the angle of curvature < 150° 2
1* Seeds curved or U-shaped, the angle of curvature ≥ 150 8
2 Number of pits in the seed coat in the middle row ≥ 30 E. orthosperma

Seed length (658–)776–854(–971) µm, width (242–)297–334(–389) µm, angle of curvature (55–)61–99(–156)° number of pits in the middle row (23–)32–38(–47), prevailing pit shape rectangular, semilunar membrane absent on the concave side of seeds.

2* Number of pits in the seed coat in the middle row <30 3
3 Length of seeds < 600 µm 4
3* Length of seeds ≥ 600 µm 5
4 Number of pits in the seed coat in the middle row <  17 E. brochonii

Seed length (365–)533–645(–813) µm, width (217–)252–276(–312) µm, angle of curvature: 26–47(–79)°, number of pits in the middle row (12–)14–15(–17), prevailing pit shape hexangular, semilunar membrane absent on the concave side of seeds.

4* Number of pits in the seed coat in the middle row ≥ 18 E. triandra

Seed length (328)467–560(700) µm, width: (158)201–231(274) µm; angle of curvature: 58–89 (136)°, number of pits in the middle row (16)20–23(28), prevailing network-shape of pits in the seed coat, semilunar membrane absent on the concave side of seeds.

5 Number of pits in the seed coat in the middle row ≤17 E. brochonii (for description see above, after line 4)
5* Number of pits in the seed coat in the middle row >17 6
6 Angle of curvature of seeds ≤60° E. alsinastrum

Seed length (708–)799–859(–950) µm, width: (230)290–330(391) µm, angle of curvature: 33–56(91)°, number of pits in the middle row: (18)21–23(26), prevailing rectangular shape of pits in the seed coat, semilunar membrane absent on the concave side of seeds.

6*Angle of curvature of seeds > 60° 7
7 Width of seeds ≥ 320 µm E. macropoda

Seed length: (568–)666–732(–830) µm, width: (282–)329–360(–407) µm, angle of curvature (78–)111–134(–167)°, number of pits in the middle row: (13–)19–23(–29); prevailing rectangular shape of pits in the seed coat, usually semilunar membrane absent on the concave side of seeds.

7* Width of seeds < 320 µm E. hexandra

Seed length: (593–)656–697(–760) µm, width: (223–)283–322(–381) µm; angle of curvation (15–) 77–118(–180)°, number of pits in the middle row: (16–)19–21(–24), the shape of pits in the seed coat hexagonal and rectangular, semilunar membrane absent on the concave side of seeds.

8 Number of pits in the seed coat in the middle row ≥ 30 9
8* Number of pits in the seed coat in the middle row <30 10
9 Length of seeds < 600 µm E. campylosperma

Seed length: (439–)505–549(–615) µm, width: (274–)419–517(–663) µm, angle of curvation: (222–)265–294(–337)°, number of pits in the middle row: (15–)31–42(–59), narrow rectangular or round shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.

9* Length of seeds ≥600 µm E.hydropiper

Seed length: (548–)602–638(–693) µm, width: (367–)454–512(–599) µm, angle of curvation: (246–)273–291(–318)°, number of pits in the middle row: (22–)37–48(–62), prevailing rectangular shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.

10 Length of seeds ≤ 600 µm E. hungarica

Seed length: (296–)459–567(–730) µm, width: (284–)357–405(–477) µm; angle of curvation: (161–)213–247(–299)°, number of pits in the middle row: (11–)20–26(–35), prevailing hexagonal shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.

10* Length of seeds >  600 µm E. gussonei

Seed length: (539–)627–685(–774) µm, width: (325–)436–509(–620) µm; angle of curvation: (80–)180–247(–347)°, number of pits in the middle row: 17–23(–32), prevailing hexagonal shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.

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