Aquatic Botany 153 (2019) 15–23 Contents lists available at ScienceDirect Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot Phenotypic variation disguises genetic differences among Najas major and N. marina, and their hybrids T Stephanie Rüegga, Christian Bräuchlerb, Juergen Geista, Günther Heublc, Arnulf Melzera, ⁎ Uta Raedera, a Aquatic Systems Biology Unit, Limnological Research Station Iffeldorf, Department of Ecology and Ecosystem Management, Technical University of Munich, Hofmark 1-3, D-82393 Iffeldorf, Germany b Restoration Ecology, Department of Ecology and Ecosystem Management, Technical University of Munich, Emil-Ramann-Str. 6, D-85354 Freising-Weihenstephan, Germany c Systematic Botany and Mycology, Department Biology I, Ludwig-Maximilians University, GeoBio Center LMU, Menzinger Strasse 67, D-80638 Munich, Germany A R T I C LE I N FO A B S T R A C T Keywords: Macrophytes Morphology ITS PCR ITS-RFLP Identification WFD mapping In this study we examined morphological variation in two macrophyte species (Najas major All., N. marina L. and their hybrids) obtained from German fresh water systems. Clear-cut delimitation of these two taxa is notoriously difficult but important as they are used as indicator organisms for water quality within the European Water Framework Directive (WFD) and have recently been revealed as two genetically separate species. To reliably identify both taxa and their hybrids, we used an integrative approach testing six discrete and two ratio-based morphological leaf and seed characteristics against restriction fragment-length polymorphism patterns (RFLP) based on PCR of rDNA internal transcribed spacer (ITS) sequences. Morphometric data of 475 plant individuals from 25 different German lakes showed basic correlation with the species delimitation suggested by molecular data, but revealed considerable overlap for characteristic state ranges, which can lead to misidentification of species if a low number of observations is made on these traits. Hybrids showed a mosaic of both parental and intermediate morphological traits. Notably, the traditionally employed feature “number of teeth along the margin on the leaf sheaths” proved to be of low diagnostic value. Leaf dimensions, especially leaf widths, were shown to be more reliable characteristics for distinguishing parental taxa. In practice, further use of both Najas species within the implementation of the WFD should be accompanied by molecular genetic testing to detect both cryptic co-occurrence and hybridization. This study points out the importance of thorough sampling and molecular screening in widespread and taxonomically difficult groups. 1. Introduction Global environmental change, degradation of natural habitats and the resulting loss in biodiversity are major anthropogenic factors affecting freshwater ecosystems (Geist, 2011). To mitigate these processes, efforts to protect and document biodiversity by applying classical taxonomic and modern molecular techniques are made (Duminil and Di Michele, 2009; Steele and Pires, 2011). However, cryptic species and hybridization often impede the identification and correct distribution mapping of plant species. This problematic phenomenon is specifically widespread within macrophytes due to their simplified anatomy (e.g. reduced leaves) and considerable morphological variability in the few diagnostic traits available to identify species. In addition, low crossing barriers result in high rates of hybridization ⁎ (Sculthorpe, 1967; Les and Philbrick, 1993). Basic molecular techniques such as DNA sequencing or amplified fragment length polymorphism (AFLP) analysis provided new approaches to detect hybridization and previously unrecognized cryptic diversity in various notoriously challenging genera of macrophytes i.e. Potamogeton (Whittall et al., 2004; Kaplan and Fehrer, 2013); Ranunculus section Batrachium (Hörandl and Emadzade, 2012; Zalewska-Gałosz et al., 2015); Najas (Les et al., 2010); Callitriche (Prančl et al., 2014), and Chara (Boegle et al., 2007). Among the problems still not readily addressed in many groups are hybrids and their confounding effect on morphological distinction of taxa (Rieseberg et al., 1993). Species misidentification and errors in delineation of their spatial distribution may have severe consequences on applied practice, especially if species are used as indicators. Within the implementation of the European Water Framework Directive (WFD) Corresponding author. E-mail address: uta.raeder@tum.de (U. Raeder). https://doi.org/10.1016/j.aquabot.2018.11.005 Received 18 June 2018; Received in revised form 6 November 2018; Accepted 10 November 2018 Available online 12 November 2018 0304-3770/ © 2018 Elsevier B.V. All rights reserved. Aquatic Botany 153 (2019) 15–23 S. Rüegg et al. identification without sequencing and using morphology. (3) To assess and discuss the consequences of our findings for further mapping procedures involving possibly cryptic macrophyte species in general and with regard to the usage of Najas within WFD procedures in particular. Najas marina and N. major should thereby serve as examples of widespread species in which thorough sampling helps to understand morphological implications of hybridization and to show how insufficient morphological data influences species detection. various aquatic macrophyte species are applied as biological control elements for ecological assessment and monitoring of water quality (Penning et al., 2008; Stelzer et al., 2005). For European freshwaters, 58 macrophyte species are listed as hybridizing (Moe et al., 2013) and approximately ten of them are indicator organisms of eutrophication within WFD guidelines for Germany (Schaumburg et al., 2014) and other European countries (Penning et al., 2008). Hybrid species frequently exhibit a mosaic of parental and intermediate characteristics (Rieseberg et al., 1993) and recognizing them in the field is further impeded by overall shrinking taxonomic expertise (Figueiredo and Smith, 2015). One of the most popular molecular markers for analyzing plant groups is the internal transcribed spacer region (ITS) of the nuclear ribosomal 18S–5.8S–26S cistron (Baldwin et al., 1995). Despite the known drawbacks for this marker (Álvarez and Wendel, 2003), its use with subsequent cloning and in combination with plastid sequence data has proven to be sufficient for tracing hybridization in multiple studies (Les et al., 2010; Kaplan and Fehrer, 2013; Tippery and Les, 2013). Although molecular methods allow and facilitate identification of hybrids and cryptic species, delimitation problems in the field prevail due to lack of comprehensive data for most critical plant groups. Detection of hybrids happens mostly incidentally and sometimes stays unrecognized until molecular data is collected and screened (Les et al., 2010; Rüegg et al., 2017). Even then, data examination has to be done carefully with respect to the chosen target region, since artefacts such as superimposed and illegible sequences can complicate recognition of their hybrid nature (Whittall et al., 2004; Tippery and Les, 2013). In this study, we use Najas major All. and N. marina L., two subcosmopolitan, annual, dioecious, submerged macrophyte species to demonstrate the possibility of re-examining and testing morphological concepts with the aid of molecular genetic techniques. Both were considered subspecies of N. marina (Viinikka, 1976) or were often merged under the Najas marina L. s.l. (see Bräuchler, 2015 for discussion). The species thereby serve as an example for a critical group with confounding taxonomy and the potential for cryptic divergence, including both taxa mentioned. Both taxa show reduced and convergent morphological traits, which exhibit broad morphological variation, that often overlap (Viinikka, 1976; Triest, 1988). This leads to persistent morphological and taxonomic confusion as well as inaccurate recording of species distribution in Europe and Germany (Lansdown, 2016; Bettinger et al., 2013). Nonetheless, several studies were able to show that the taxa are differentiated in their karyotype (Viinikka, 1976) and isozyme patterns (Triest et al., 1986) and molecular data suggest treating them as separate species (Rüegg et al., 2017). Due to phenological differences, the two taxa have been shown to be able to hybridize so far only unidirectionally when male N. marina pollinate female N. major plants, which results in the formation of mostly infertile offspring (Viinikka, 1976; Triest, 1989). Only a few naturally occurring hybrids have been identified in Europe (Triest, 1989), but for given reasons many more may have remained undetected. The possibility of hybrid formation and overlap of morphological variation is obscuring the accurate identification of species in the field, and is limiting utility of both taxa as currently distinct indicator organisms according to the requirements of the WFD for German lakes (Schaumburg et al., 2004, 2014). This emphasizes the need for an integrative approach using a combination of multiple independent sets of characteristics (Duminil and Di Michele, 2009) including molecular and morphological data, in order to facilitate proper species identification in this problematic taxon not only for assessing actual specific spread but also for planning conservation and/or management strategies. The core objectives of our study were to (1) overcome persisting identification problems by testing diagnostic morphological characteristics on a genetic background including parent species and hybrids and to compare these results to measurements known from literature. (2) To develop a simple and rapid PCR‐RFLP method for monitoring genetic variation using the ITS marker region that allows an accurate species 2. Materials and methods 2.1. Study area and sampling strategy A total of 475 adult and flowering plant individuals were sampled from populations of N. marina and N. major in 25 lakes throughout Germany (Appendix A). All specimens were collected by diving along a point or strip transect from August to October. 315 plants were collected as described in 2010 (125 from N. major, 190 from N. marina), 160 samples were gathered in 2012 and 2015 (71 from N. major, from 76 N. marina, and 13 hybrids). In several cases, multiple transects per lake were sampled (Appendix A). Depending on density of the plant stands, three to ten individuals per transect were collected. One representative DNA sample and voucher specimen was prepared per transect for the survey in 2010, whereas DNA samples and voucher specimen were taken from each individual collected in 2012 and 2015 (Appendix A). All vouchered individuals were analyzed molecularly as described in Rüegg et al. (2017). Morphological measurements were taken from all individuals collected throughout these years. Herbarium vouchers were deposited at TUM herbarium (Thiers, 2017) in the macrophyte reference collection at the Limnological Research Station Iffeldorf. Of the sites previously described (Rüegg et al., 2017), 17 plants were collected at Lake Abtsdorf in 2015 as a reference for N. major, 50 plants were obtained from Lakes Starnberg (38) in 2012 and Lake Constance (12) in 2015, as a reference for N. marina. All three lakes show minimum risk of introduction of the other species from neighboring lakes due to the distance among them and to the next lakes housing the respective other species. In Lake Staffelsee, identified as hybrid zone in our previous study (Rüegg et al., 2017), 110 individuals of both taxa and their hybrids (54 from N. major, 26 from N. marina, and 13 hybrids) were collected in 2012 and 2015 at the same locations using GPS devices and data points. Standard taxonomic keys were used to identify plants prior to further morphological and molecular analysis (Casper and Krausch, 1980; Van de Weyer et al., 2011). 2.2. Morphometrics For this study a total number of 1737 leaves was analyzed: N. major n = 724, N. marina n = 948, hybrids n = 65. Quantitative morphological characteristics were measured from 475 plants (N. major n = 196, N. marina n = 266, hybrids n = 13) and 408 seeds (N. major n = 159, N. marina n = 239, hybrids n = 10) using a dissecting microscope (6.5–40·× magnification) linked to a digital camera (Kappa PS20 H), which was controlled by interactive software (Kappa imageBASE v2.7). In a preliminary study from 2010, 8–10 plants per transect were collected and leaf measurements were taken from three leaves per plant by hand with the aid of graph paper. Overall, 315 plants (N. major n = 125, N. marina n = 190, number of plants included in the measurements mentioned above) were measured that way. From 160 plants that were collected in 2012 and 2015 (N. major n = 71; N. marina n = 76, hybrids = 13), three to five representative leaves and five seeds (when present) per individual were randomly chosen for measurements. Characteristics examined were: length and width of seed (SL and SW) as well as leaf length (LL) and two different widths on each leaf: one width at teeth (= leaf widths broad: LWB) and at sinuses (= leaf widths narrow: LWN), all measured in mm. Each leaf width was measured at up to three points, depending on leaf length. LWB and LWN 16 Aquatic Botany 153 (2019) 15–23 S. Rüegg et al. (bonferroni). Linear Discriminant Analysis (LDA) and Canonical Variate Analysis (CVA) was conducted on quantitative morphological data to determine which characteristics best discriminate the studied species using the functions lda with default settings (package MASS) or the function CVAbipl.pred.regions (package UBbipl). The CVA is a form of multivariate analysis, which minimizes within group (replicate) variation and maximizes the between-group variance (Gower et al., 2010). Afterward, a leave-one-out cross-validation (loocv) method was applied to validate the models and calculate error rates for reclassification. Plots were generated using the ggplot2 or ggpubr package. were both measured vertical to the midrib of the leaf. For both measuring methods (graph paper and digital), fresh leaves were prepared equally by placing them between two microscopic glass slides. Moreover, the total number of marginal teeth on the leaf sheath was noted for each leaf. Though this characteristic was considered to be of no true diagnostic value (Viinikka, 1976; Triest, 1988), its perpetuated employment within recent taxonomic keys (Casper and Krausch, 1980; Van de Weyer et al., 2011) emphasizes the need of a thorough re-examination. For each plant, sex was determined as a non-quantitative characteristic. Due to seasonal sex ratio patterns (Hoffmann et al., 2014) and the late sampling dates, more female individuals (89%) were collected, though sex-related differences were not significant for any of the traits examined (Wilcox test, p > 0.05). 3. Results 3.1. PCR - RFLP analysis 2.3. PCR – RFLP analysis RFLP analysis using Hind III showed one undigested band at approximately 750 bp for N. major samples whereas the digestion of N. marina resulted in two fragments shown as two bands at approximately 475 bp and 270 bp in the agarose gel. A molecular substitution from C to A at site 284 within the ITS2 region causes a loss of the Hind III recognition site in N. major. The hybrid nature of 13 samples could be confirmed by additive RFLP banding patterns from both parents showing a combination of all three bands. The identity of various hybrid specimens (Appendix A) was previously confirmed by cloning and sequencing of the ITS region, resulting in the presence of both parental sequences as reported in Rüegg et al. (2017). All 34 samples of N. major displayed the described RFLP pattern, though two samples had to be reexamined due to possible contamination. For N. marina specimens 38 samples showed the expected pattern. RFLP analysis results revealing hybrids by additive RFLP banding patterns was confirmed by a second analysis for those 13 samples. Restriction site analysis of PCR amplified ribosomal ITS fragments was performed for a representative number of both taxa and included samples collected for a previous study (Rüegg et al., 2017) in order to verify type of specimens characterized earlier by genetic or morphological analysis (see Appendix A). RFLP analysis was performed overall on 35 specimens of N. major, 38 specimens of N. marina, and 11 hybrids. For each individual, DNA extraction and PCR amplification were carried out using the primer pair leu1 (Vargas et al., 1998) and its4 (White et al., 1990). Purification of products and cloning followed by sequencing was performed as reported by Rüegg et al. (2017). Between 1- 4 μL of the purified PCR products (1–20 ng/μL; 0.1–1.5 nM) were digested overnight (app. 16 h) in a total volume of 20 μL containing 2 μL, of enzyme (1 U/ μL) (Thermo Fisher Scientific, Massachusetts, USA), 2 μL of 10x BSA Buffer R that is provided with the enzyme (1 mM Tris HCl; 10 mM KCl; 0.02 mg/mL BSA, 0.1 mM EDTA, 0.1 mM DTT) and bdH2O. Hind III was chosen as a restriction enzyme based on the restrictions map tool implemented in BioEdit (Hall, 1999) and double checked with the virtual digestion tool available online at restrictionmapper.org. Hind III cuts once at position 278 (5′ A↓AGT_T 3′) within the ITS2 of the reference sequence of N. marina (KT596460) but not in N. major. Therefore, we expected the method to allow for a distinction between the two taxa and their hybrids, which consequently could be identified by undigested PCR products (one band) and digested DNA fragments (two bands). Digestion efficiency and length of resulting fragments were checked on a 2% (w/v) agarose gel, using ethidium bromide staining and a 100 kb ladder (Thermo Fisher Scientific, Massachusetts, USA). Doubtful RFLP results were repeated and double-checked. 3.2. Monitoring and identification of plants PCR - RFLP helped in delimitation of 13 hybrid specimens, which would have been otherwise identified as N. major based on morphology (Tables 1 and 2). By identifying plant material genetically, it was shown that hybrids occur naturally in mixed populations at Lake Staffelsee. Due to higher sampling density and a better understanding of co-occurrence of the two different taxa, 13 hybrid specimens were collected from Lake Staffelsee in two different years, 2012 and 2015. General occurrence of hybrid individuals was persistent, but their frequency differed depending on the abundance and mixed growth of parental taxa. Apparently, F1 hybrids develop anew each year, since no fertile seeds have been reported or detected on hybrid individuals so far. Nonetheless, we chose to include hybrid seeds in this study since they 2.4. Data analysis Statistical analysis of cumulative data was compiled using the open source software R v. 3.4.4 (R Development Core Team, 2013). Taxa were identified based on genetic markers (PCR-RFLP) as described before and in all statistical analyses, one grouping variable (taxa) was used. Due to the nested design of the study (multiple measurements per leaf and individual, randomly chosen leaves and plants) standard deviation, standard error, and means were calculated for each individual. Means were then pooled over the two different taxa and their hybrids for further analysis. Number of plants or individuals is given for each statistic. The different datasets where inspected for normality of residuals (shapiro.test) and homogeneity of variances (var.test) via the given functions and diagnostic plots. If values were not normally distributed, non-parametric tests were conducted (wilcox.test). Otherwise, an ANOVA (aov function) with default setting was performed. Tukey’s HSD was conducted as posthoc test for unequal sample frequencies to assess group specific differences between means of quantitative characteristics. Count data (number of teeth) were tested using a generalized linear model (glm, family = poisson (link = "log")) followed by a multiple comparison (package multcomp) with adjusted p values Table 1 Mean value, standard deviation (SD) and standard error (SE) for morphological characteristics measured (n is the number of leaves or seeds measured; leaves: N. marina, n = 725, N. major, n = 948, hybrids, n = 64; seeds: N. major, n = 159 N. marina, n = 219, hybrids, n = 10). Species Statistics LWB leaf width broad (mm) LWN leaf width narrow (mm) Leaf length (mm) Number of teeth on margin of leaf Seed width (mm) Seed length (mm) N. major All. Mean SD SE Mean SD SE Mean SD SE 4.83 0.91 0.03 2.54 0.83 0.03 4.10 0.68 0.08 1.82 0.37 0.01 0.92 0.30 0.01 1.44 0.25 0.03 21.20 5.31 0.19 18.37 6.91 0.22 16.55 3.23 0.40 1.87 1.22 0.05 3.01 1.29 0.04 2.57 1.27 0.27 2.35 0.40 0.03 1.57 0.31 0.02 2.47 0.85 0.20 4.79 0.54 0.04 4.00 0.34 0.02 5.11 0.62 0.18 N. marina L. Hybrids 17 Aquatic Botany 153 (2019) 15–23 S. Rüegg et al. Table 2 Measurements of leaf and seed characteristics in mm from literature and calculated in this study (Table 1). Values are given as mean values ± SD. Full ranges of observed traits are given in brackets. All measurements were taken from fresh plant material. Citation index: 1) Viinikka (1976), 2) Casper and Krausch (1980), 3) Triest et al. (1986), in studies 1) and 3) dried leaves were measured. Character Najas major All. Najas marina L. Hybrids Citation Leaf width (mm) broad (LWB) narrow (LWN) Leaf length (mm) (2.7 -) 3.5 - 5.1 (- 6.1) (0.8 -) 1.0 - 1.5 (- 2.5) (2.2 -) 3.9 - 5.7 (- 8.0) (0.6 -) 1.5 - 2.2 (- 3.0) (10 -) 19 - 34 (- 45) (11.6 -) 15.1 - 23.3 (- 28.9) (8.5 -) 15.9 - 26.5 (- 44.0) Without, rarely one (0 -) 1 - 3 (- 6) (3.5 -) 4.5 - 6.4 (-8.0) (3.3 -) 4.2 - 5.4 (-6.6) (3.4 -) 4.2 - 5.3 (- 6.6) (2.7 -) 3.5 - 5.1 (-6.1) (1.3 -) 2.1 - 2.8 (- 4.1) (1.5 -) 1.9 -2.7 (- 3.4) (1.5 -) 1.8 - 2.8 (- 3.3) (0.2 -) 0.5 - 0.9 (- 1.1) (0.9 -) 1.7 - 3.4 (- 5.5) (0.4 -) 0.7 - 1.1 (- 2.8) (4 -) 9 - 26 (- 38) (4.4 -) 6.5 - 11.1 (- 12.0) (4.4 -) 11.3 - 24.7 (- 42.0) 1 - 3 (4) on each side (0 -) 2 - 4 (- 8) (2.3 -) 3.0 - 4.0 (- 4.8) (2.6 -) 3.4 - 4.2 (- 5.1) (2.9 -) 3.7 - 4.3 (- 5.0) (1.5 -) 1.8 - 2.8 (- 3.3) (0.9 -) 1.2 - 2.0 (- 2.7) (0.9 -) 1.3 - 1.9 (- 2.5) (2.1 -) 3.4 - 4.8 (- 5.7) (0.9 -) 1.1 - 1.7 (-2.0) 3) 1) + 2) This study (10.1 -) 13.3 - 19.8 (- 24.1) 1) + 2) 3) This study 2) This study 1) + 2) 3) This study 1) 3) This study Number of teeth on margin of leaf sheath Seed length (mm) Seed width (mm) Cross validation error rate Associated characteristics 1 1 2 3 4 0.129 0.175 0.129 0.103 0.092 LWN (leaf width narrow) LWB (leaf width broad) LWN + LWB LWN + LWB + teeth LWN + LWB + teeth + length (1.3 -) 1.6 - 3.3 (- 3.9) significant difference could be detected between N. major and hybrid individuals (Wilxoc test p = 0.181), nor between N. marina and hybrids (Wilcox-test p = 0.438). Seed width (Tukey test: p = 0.778), seed length (Tukey test: p = 0.450) and seed ratio length: width (Tukey test: p = 0.834) did not show any significant differences between N. major and hybrids either. N. marina and hybrid seed differed significantly from each other in width and length (Tukey test: p < 0.001), but not in ratios of seed length: width (Tukey test: p = 0.187) (Fig. 3c). Notably, only five seeds each could be measured from two of the overall 13 as hybrids-identified individuals. LDA of four quantitative morphological characteristics indicated good separation between N. major and N. marina (Figs. 4a,b). The first linear discriminant LD1 explains more than 99% of the between-group variance. From all four morphological characteristics (LWN, LWB, length and teeth) used in the model, the variables that provided most effective discrimination between N. marina and N. major were LWN (coefficient 2.636) and LBW (coefficient 0.709). The number of teeth on the leaf margins and leaf length showed weaker influence on the first Linear Discriminant LD1 (teeth: coefficient -0.3993, length: coefficient 0.0337) and therefore do not exert such a strong influence on the discrimination of the taxa and their hybrids. Values for hybrid samples show overlap with N. major samples, but are for the most part located between the two taxa (Fig. 4b). The CVA misclassified more individuals from N. major than from N. marina. Sample means for individuals from N. major show broader ranges for measurements of leaf widths and the number of teeth, whereas sample means from N. marina plants varied more in leaf length. Both plots (LDA and CVA) show that hybrid samples are intermediate between both parental taxa but overlap more with N. major than with N. marina (Fig. 4a,b). Four N. major plants are misclassified according to CVA analysis as N. marina, and were drawn from Lakes Staffelsee, Mindelsee and Muttelsee. Eight samples of N. marina that are misclassified as hybrids and lie within the 90% bag of N. major samples were obtained from Lakes Staffelsee, Starnberg, Wörthsee and Pelham. All of these lakes lie within sympatric ranges, except for Lake Starnberg and Lake Wörthsee. Table 3 Smallest CVA cross validation error rates calculated using the leave-one-out (loocv) method for different sized subsets of the characteristics used to describe the different taxa and their hybrids. Mean values over the individuals are used calculating the error rate. Subset size (0 -) 1 - 4 (- 6) did not show any signs of deformation or abnormal growth. 3.3. Morphological variation The results of the morphometric measurements on leaves and seeds demonstrated high variability within each taxon, depicted also by a high number of outliers (Figs. 1–3b, c). Character state ranges measured in this study showed considerable overlap with interquartile ranges for both taxa and their hybrids (Table 1, Fig. 1). Standard deviations and errors were relatively low due to high number of measurements and accuracy of tools used (Table 1). Significant among-group differences between N. marina and N. major were calculated for mean values pooled from individuals for almost all leaf and seed characters measured using ANOVA: LWN (F2, 472 = 843, p < 0.001), LWB (F2, 472 = 696, p < 0.001), and length (F2, 472 = 13.82, p < 0.001), as well as seed width (F 2/72 = 69.31; p < 0.001), seed length (F 2/80 = 52.99; p < 0.001) and ratio length: width (F 2/75 = 29; p < 0.001). Only the characteristic ratio narrow: broad did not obtain such high amonggroup difference (F2, 471 = 0.337, p = 0.71), which could also be seen in comparing mean values (as described below). LWN and LWB were shown to correlate highly with each other (function rcorr 0.94). Mean values of all traits measured showed significant differences between N. marina and N. major (Tukey test p ≤ 0.001) but not between each of them and the hybrids, depending upon trait (Fig. 1c, d). With regard to LWN and LWB, hybrids did also differ significantly from the two taxa (Tukey test p < 0.001). Leaf widths (LWN, LWB) measured from the 13 hybrid plants showed to be intermediate between the parental taxa (Table 1). Measurements taken from hybrid seeds otherwise, showed closer resemblance to characteristic states taken for N. major plants (Figs. 1a–c; 3; Table 1). Except for the traits of leaf length (Tukey test p = 0.256) and number of the teeth on leaf sheaths (Tukey test p < 0.038), hybrid individuals are more likely to appear like N. marina plants (Figs. 1d; 2b). For the trait leaf ratio narrow: broad no 4. Discussion 4.1. Identification of taxa and their hybrids Samples of N. major and N. marina could be accurately discriminated based on molecular markers and afterwards morphological traits were critically verified within and among molecularly defined groups. Morphological results are in accordance with measurements for karyotype A and B conducted in studies by Viinikka (1976) and Triest 18 Aquatic Botany 153 (2019) 15–23 S. Rüegg et al. Fig. 1. (a) – (d) Leaf characteristics among N. major (n is the number of leaves measured, n = 724), N. marina (n = 948), and their hybrids (n = 65). Boxplots indicate interquartile ranges with median values (heavy lines), “whiskers” are extended to a maximum of 1.5 × interquartile range, and outliers are shown as black circles. Notches were added approximating a 95% confidence interval (CI) for the median. Comparison of mean values for the characteristics were pooled over individuals: N. major n = 196, N. marina n = 266, hybrids n = 13. Characteristic LWN = leaf width narrow (a) and LWB = width broad (b) differed significantly between N. major (blue boxes), N. marina (yellow boxes), and their hybrids (gray boxes). No differences were detected between parental taxa and hybrids in the characteristic leaf width ratio narrow: broad (c) between N. major and hybrids nor between N. marina and hybrids For N. major and hybrids, mean values of the characteristic leaf length (d) showed significant differences (Tukey test: p = 0.006), but mean values of N. marina and hybrid plants did not (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). Fig. 2. (a) Histogram showing the counts for the number of teeth on the leaf sheaths for different taxa and the hybrids: N. major (n is the number of leaves measured, n = 725), N. marina (n = 948) and hybrids (n = 64). Highest number of counts for teeth on the leaf sheaths was observed for both species N. major (blue bars) and N. marina (yellow bars) at “2″. No teeth (0) were observed on leaf sheaths form 69 leaves from 47 different N. major plants but were also observed on 6 leaves from 6 different N. marina plants, and on five leaves of three different hybrid plants (gray bars) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). (b) Violin plot for the characteristic “number of teeth on the leaf sheaths” shown for different taxa and the hybrids: N. major (n is the number of plants measured, n = 196), N. marina (n = 266) and hybrids (n = 13). Significant differences for the characteristic number of teeth on the leaf sheaths were observed between N. marina (yellow violin) and N. major (blue violin), N. major and hybrids (gray violin) but not between N. marina and hybrids. Plots show the density or distribution shape of the data. The box inside the violin indicates the interquartile ranges with mean values (heavy lines, ‘whiskers’ = 1.5 × interquartile range). 19 Aquatic Botany 153 (2019) 15–23 S. Rüegg et al. Fig. 3. (a) – (c) Seed characteristics among different taxa and their hybrids. N. major (n is the number of seeds measured = 159), N. marina (n = 219) and hybrids (n = 10). Taxa were delimited based on distinct RFLP ITS patterns. Boxplots indicate interquartile ranges with median values (heavy lines), ‘whiskers’ are extended to a maximum of 1.5 × interquartile range, and outliers are shown as black circles. Notches were added approximating a 95% confidence interval (CI) for the median. Notches are outside for hybrids due to low sample size. Mean values pooled over individuals (n) differed for all characteristics significantly between N. major (blue boxes, n = 32) and N. marina (n = 49, yellow boxes) (Tukey test: p < 0.001). For N. major and hybrids (n = 2, gray boxes) mean values did not differ significantly for any characteristic tested, whereas N. marina seeds differed significantly from hybrid seeds with regard to width (a) and length (b) but did not differ in ratios of seed length: width (c). N. major seeds were generally “bigger” than those from N. marina, though showing a lower length to width ratio. Hybrid seed appear morphological like N. major seeds (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). spines/teeth on the margins of the leaf sheaths, as is done in most standard keys for final delimitation of the two taxa. Measurements of leaf widths as given in Table 1 should be used and included in those keys instead. In cases of doubt or a high probability that hybrids are present, only molecular analysis will help to make a clear-cut decision. The integrative approach used in this study to distinguish between the two Najas taxa and their hybrids can be considered successful because we gained a more accurate understanding of the distribution patterns of N. marina and N. major, identified reasons for misidentification of plants, and proposed a fast method for clear cut identification. Morphology-based identification methods can be useful if several characteristics are combined, as shown in this study, although they reach their limits because measurements are time consuming and have to be made in sufficiently great numbers and detail using digital imaging. In the case of Najas, prior recognition of the presence of cryptic species within various regional databases (Bettinger et al., 2013) would have been advantageous also for other plant surveys e.g. Hoffmann and Raeder (2016). The correction of geographic range maps should be considered, and accurate information should be given for distribution of N. marina and N. major underlining sympatric ranges. Further expansions of (sympatric) ranges for both taxa seem very likely due to the supposed cryptic (Rüegg et al., 2017) and invasive spread reported for some German lakes (Hoffmann and Raeder, 2016). The development of a PCR-based RFLP method for identification is considered useful and can be recommended as a quicker and cheaper alternative to DNA barcoding by sequencing and cloning of doubtful sample material in Najas. Molecular tools like DNA barcoding should be used with caution and in conjunction with other methods (Duminil and Di Michele, 2009; Steele and Pires, 2011). Nevertheless, these methods helped substantially in uncovering enduring mistakes and have already revealed cryptic introductions or invasions for other aquatic species (Whittall et al., 2004; Les et al., 2013). Only by generating the molecular datasets is was possible to detect hybrids and reliably assesses the distribution of taxa in this study, but linkage to multiple morphological characteristics that can be measured and documented readily is required to re-evaluate and overcome persistent identification problems. Combined approaches will aid in establishing reliable molecular (1988) (Table 2). Both N. marina and N. major used in this and previous studies correspond thereby not only genetically (Rüegg et al., 2017) but also morphologically to karyotypes A and B. Detection of hybrids as well as a more detailed morphological characterization of those plants was only possible based on molecular results. The sole morphological identification of hybrid plants is almost impossible in the field because different quantitative traits of hybrids in this and other studies (Triest, 1989) appeared either phenotypically intermediate or resembled one of the parents. In this study leaf measurements, (LWB and LWN) of hybrids are intermediate between both parental taxa whereas seed measurements of hybrids resemble rather N. major plants. Some hybrid traits like teeth on the leaf sheaths, leaf or seed ratios were mosaically distributed between both taxa. Other aquatic plant hybrids like Nymphaea are also known for such limitations and representing a mosaic of both parental and intermediate characters rather than strictly intermediate ones (Les et al., 2004; Rieseberg et al., 1993). Another example for the concealment of hybrids in Najas was described by Les et al. (2010), where identification of N. flexilis × N. guadalupensis subsp. olivacea hybrids was hampered by their strong resemblance to one of the parental plant species (N. guadalupensis). Only certain leaf (i.e. LWN) and mainly seed (i.e. SW and SL) characteristics could be shown to correlate with genetic types and can be considered useful for a morphological distinction between the two taxa (without hybrids). Results are in accordance with a previous study by Peredo et al. (2011), who arrived at the same conclusion that N. marina can be regarded as an “aggregate taxon of two cryptic species”. Except for minor seed characters, no consistent pattern of morphological variations could be detected to delimit infraspecific taxa reliably. However, the applicability of seed for delimitation of the two taxa is limited by their availability due to dioecy of plants as well as sampling date and can therefore only be used as additional characteristics when present. Further problematic vegetative characteristics are the length of leaves and the number of teeth on the leaf sheaths, which were already considered of low diagnostic value by Triest et al. (1986). Viinikka (1976) suggested that peculiarity of teeth and the length of leaves is closely dependent on the stage of development due to their slow growth. Based on our results, we strongly recommend not using just the 20 Aquatic Botany 153 (2019) 15–23 S. Rüegg et al. Fig. 4. (a) Biplot of the canonical variate analysis (CVA) of all samples. N. major (n = number of plant individuals = 196), N. marina (n = 265), and hybrids (n = 13) using all four morphological variables (LWN = width_wo, LWB = width_w, length and teeth). Classification regions were added (shown as background colors) and 0.9 bags were drawn. A bag approximates the box in a boxplot, where 90% of the data points lie within the polygon. The amount of separation obtained between the species is shown using the biplot as a graphical display in the classification process. Samples that were misclassified (symbols that are not within their corresponding classification region in the plot) were all obtained from different lakes. Misclassification rates according to the CVA analysis are given in Table 3. (b) Plot of the linear discriminant analysis (LDA) of the same datasets as shown in (a). The first discriminant axis (LD1) separates the taxa including hybrids describing 99.82% of the variation expressed in the data. The second discriminant axis (LD2) contributes 0.18% of the variation to further distinguish the taxa and their hybrids from each other. Eggstätt-Hemhofer Lake district (all in Bavaria; Rüegg et al. (2017)), Lakes Weutschsee and Oberucker (both in Mecklenburg-Pommerania; Doll and Pankow (1989)), Lakes Nemitz and Tegel (Brandenburg and Berlin; Viinikka (1976)). Besides the restriction of co-occurrence of the species based on abiotic factors such as biogeographic history, also biotic factors such as temperature can be assumed to play a key role for the hybridization of both taxa. The life cycle of Najas plants is known to depend heavily on temperature, by influencing germination (Handley and Davy, 2005) as well as florescence. Triest (1991) reported that differences in flowering time form a hybridization barrier in populations observed in the Swiss Alp region. Gender-related differences in flowering times for N. marina plants were also described in southern German regions (Hoffmann et al., 2014). Formation of unidirectional hybrids resulting in hybrid plants that partly resemble N. major plants seems plausible for hybrid markers to identify plants with reduced morphology and high phenotypic plasticity based on thorough taxonomic work, especially when disagreement between both traditional morphological and molecular methods still prevails (Duminil et al., 2009). 4.2. Ecology of taxa and their hybrids -: implications and recommendations for further mapping and studies Hybrids of the two Najas taxa are expected to appear spontaneously in other lakes as well, since both taxa propagate each year exclusively by sexual reproduction via underwater pollination (Triest, 1988). The only molecularly verified hybrids so far have been proven to exist in Lakes Sempach and Pfäffikon, located in Switzerland (Triest, 1989), but more lakes located in Germany with the co-occurrence of both taxa are already known, e.g. Lake Waging-Taching and some lakes of the 21 Aquatic Botany 153 (2019) 15–23 S. Rüegg et al. (Nguyen et al., 2014). A list of aquatic species from taxonomic and morphologically confounding groups should be developed, thus emphasizing the need of molecular identification. Furthermore, a herbarium reference collection of problematic taxa should be established for each lake, e.g. in the course of regular monitoring according to WFD guidelines. Species selection should be based on their importance as indicators, their vulnerability status, or their role as invasive species in order to facilitate identification and further decision making. specimens detected in this study. Future investigations should determine whether lakes with both taxa and hybrids have significantly different temperature profiles in comparison to lakes containing only one of the two taxa. Sufficient sites exist to pursue additional ecological, morphological and molecular investigations. Since misidentification of plants from both taxa is most likely to happen in areas with sympatric distribution, the detection and mapping of co-occurrence and spread of either taxa is more important in the course of WFD related monitoring than simply confirming the identity of doubtful specimens and potential hybrids with the aid of molecular methods. Thresholds of measurements of leaf characteristics as given in this study (Table 2) can be useful for assessing if one of the taxa is present in a lake, but have to be applied carefully and in sufficiently high numbers. We also recommend deriving multiple measurement from distinct plants and various leaves as done in this study. Highly experienced taxonomists may be able to delimitate specimens accurately but this expertise is currently very scarce (Figueiredo and Smith, 2015). Training for mapping procedures in the course of WFD-related monitoring is time consuming and difficult, and availability of verified herbarium material is limited. By bundling all the plant material from one mapping season to process them together is one way to achieve more accurate and faster species and hybrid delimitation. This approach is surely not necessary for all species, but may be useful for those involving taxonomically problematic groups. Since we cannot solve taxonomic differentiation problems in the field with the aid of molecular methods at present, vouchering of plants as herbarium material becomes crucial, which should be done routinely during mapping, and should not create any additional expense. A consequence of not recognizing species richness or sympatric distribution in macrophyte surveys can be the misinterpretation of ecological states when confounding species are used as biological indicators, as it is the case for the two Najas taxa. In general, macrophyte surveys evaluating European water quality are based on identification of individual species and uncertainty meassurements are more sensitive when quantitative data is collected (Dudley et al., 2013). Hybrids are rarely included in assessing macrophyte species richness (Rørslett, 1991) because they are overlooked and their identifications requires molecular tools (Kaplan and Fehrer, 2013). In consequence, hybrids are not recorded at all in areas with sympatric co-occurrence of widespread species like N. major and N. marina within regular, morphologically based macrophyte surveys. Our results show that under natural conditions, hybridization between taxa in places of co-occurrence is common and hybrid origin was able to be confirmed for 13 out of the 90 plants collected (∼14%). The occurrence of hybrid plants in Lake Staffelsee was verified for two vegetative periods in 2012 and 2015. In general, it is still unclear how common and stable hybrid populations are because thorough molecular screening as done in this study is not part of the standard WFD mapping procedures. Apparently, co-occurrence of taxa and their hybrids is sustained and hybrid sterility is undoubtedly a function of the extensively rearranged genomes of the two taxa (Viinikka, 1976; Peredo et al., 2011, D. Les pers. com.). This assumption raises more questions on the hybrids ecological and evolutionary significance and should be addressed in future studies. Acknowledgments The funding of this project was provided by Bavarian State Ministry of the Environment and Consumer Protection (Grant Number TLK01U60031). The authors thank the following people for contributing to this study: Tanja Ernst for excellent technical assistance, Gerd Welzl for giving valuable advice on statistics, all scientific divers (in first place Maximiliane Schümann and Kristin Wutz), assisting students, and colleagues from the Limnological Research Station Iffeldorf, TU Munich for their various contributions. Finally, the authors appreciate the constructive feedback from our reviewers, Prof. Dr. Donald H. Les, Dr. Markus Heinrichs and Prof. Dr. Tanja Gschlößl for very helpful comments on the manuscript. References Álvarez, I., Wendel, J.F., 2003. Ribosomal ITS sequences and plant phylogenetic inference. Mol. Phylogenet. 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