Research Article |
Corresponding author: Jacek Urbaniak ( jacek.urbaniak@upwr.edu.pl ) Academic editor: Regine Jahn
© 2019 Jacek Urbaniak, Paweł Kwiatkowski.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Urbaniak J, Kwiatkowski P (2019) Taxonomic studies on the Chara section Hartmania in Poland based on morphological and molecular data. PhytoKeys 135: 71-90. https://doi.org/10.3897/phytokeys.135.36714
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Charophytes are aquatic green macroalgae, which inhabit fresh and brackish water ecosystems. In this study, four species belonging to the genus Chara were examined to determine their taxonomic status. Morphological characteristics of the plant bodies as well as plastid psaB barcoding genes were applied to test the relations among Chara species. Plants were initially classified using morphological features into four species: C. baltica, C. hispida, C. polyacantha and C. rudis, and twelve quantitative characters were used in a principal component analysis and discriminant analysis to determine groupings among the species and to determine the morphological features that best separated the groups. In the component analysis and discriminant analysis, results showed that only C. polyacantha and partly C. baltica formed separate groups. The other species C. hispida and C. rudis were only partially distinguishable. All species from one molecular group, and no differentiation in the psaB variability between them has been found.
Chara, morphological characters, species identification, taxonomy, Poland
Macroscopic algae from the genus Chara L. can be commonly found in various water bodies, such as shallow lakes, artificial ponds, slowly running waters or drainage canals. The taxonomy of the genus Chara, as well as the other representatives of the Characeae family is not easy, mostly due to the overlapping of morphological features of individual specimens belonging to different species (
Taxonomic studies based on charophyte morphology started at the end of the 19th century, and during this initial phase, many people tried to characterize the degree of morphological variation in charophytes and find traits to circumscribe distinct species. Traditionally, in the genus Chara, a narrow species concept has been used resulting in about 45 European species (
Specimens studied with GenBank accession numbers and collection sequences used in study.
Species | psaB GenBank accession number/locality | Geographical coordinates |
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C. baltica Bruz. | KX791851/ Puck, Poland | 54°42'14.09"N, 18°27'40.70"E |
KX791852/ Swarzewo, Poland | 54°45'25.19"N, 18°24'33.91"E | |
KX791853/ Rewa, Poland | 54°38'11.17"N, 18°30'37.50"E | |
C. hispida L. | KX791854/ Lake Czarne, Poland | 54°00'57.75"N, 22°59'40.22"E |
KX791855/ Lake Mikaszewo, Poland | 53°53'15.96"N, 23°21'22.84"E | |
KX791856/ Lake Białe, Poland | 53°52'03.95"N, 23°02'16.17"E | |
KX791857/ Lake Czajcze, Poland | 54°06'56.00"N, 22°28'19.44"E | |
KX791858/ Lake Wielkie, Poland | 53°20'43.74"N, 22°55'58.36"E | |
KX791859/ Lake Wigry, Poland | 54°00'34.76"N, 23°02'16.89"E | |
KX791860/ Lake Mamry, Poland | 54°06'24.87"N, 21°46'13.13"E | |
KX791861/ Lake Pobłądzie, Poland | 54°18'24.30"N, 22°45'25.25"E | |
KX791862/ Lake Muliste, Poland | 53°54'11.23"N, 23°16'15.58"E | |
KX791863/ Lake Staw, Poland | 54°01'14.44"N, 22°59'26.91"E | |
KX791864/ Lake Jeziorak, Poland | 53°43'06.51"N, 19°36'02.98"E | |
KX791865/ Lake Śniardwy, Poland | 53°47'25.95"N, 21°44'14.71"E | |
C. polyacantha A. Br. | 216/KX791866/ Lake Jasne, Poland | 54°07'56.82"N, 22°58'41.09"E |
217/KX791867/ Lake Bilskie, Poland | 54°05'03.47"N, 23°05'31.62"E | |
218/KX791868/ Lake Wigry, Poland | 54°01'48.15"N, 28°08'30.31"E | |
219/KX791869/ Lake Kockie, Poland | 53°59'40.25"N, 20°51'25.79"E | |
C. rudis Leonh. | 220/KX791870/ Lake Staw Wielki, Poland | 53°57'01.46"N, 23°08'42.92"E |
221/KX791871/ Lake Oleckie, Poland | 54°03'23.86"N, 22°30'20.63"E | |
222/KX791872/ Lake Małe, Poland | 54°03'24.09"N, 22°42'09.85"E | |
223/KX791873/ Lake Korzęckie, Poland | 54°13'29.08"N, 22°34'05.47"E | |
C. vulgaris L. | DQ229107/Poland | – |
N. axiliformis | AB191785/Japan | – |
N. pseudoflabellata | AB191766/Japan | – |
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Chara baltica Bruz. | Chara hispida var. baltica f. baltica | Chara baltica (Hartman) Bruz. |
Chara hispida var. baltica f. liljebladi | ||
Chara hispida L. | Chara hispida var. major f. major | Chara hispida L. |
Chara hispida var. hispida f. hispida | ||
Chara polyacantha A. Braun | Chara hispida var. hispida f. polyacantha | Chara aculeolata Kütz. |
Chara rudis (A. Braun) Leonh. | Chara hispida var. major f. rudis | Chara subspinosa Rupr. |
Unfortunately, previous studies of oospore morphology, oospore wall ornamentation (scanning electron microscopy, SEM) and molecular fingerprinting data did not give satisfactory results in delimitating Chara species from the section Hartmania (
In addition, the DNA barcoding method has been proposed as an alternative method for identifying taxonomic relationships in species of the Characeae family. This method can be used successfully to facilitate biodiversity and taxonomic studies of various plants (
The presented study focuses on four of the most problematic freshwater species (two diplostichous aulacanthous species, C. hispida and C. rudis, one diplostichous thylacanthous species: C. polyacantha and C. baltica as representatives of brackish water species (a diplostichous thylacanthous species, in transition to slightly isostichous). All of them belong to the section Hartmania. We applied the plastid psaB gene as well as morphological observation to test whether the distribution of haplotypes among species is in agreement with the species delimitation.
The plants were collected manually or using a hook directly from the field. We collected mature specimens and determined according to the
We have used the charophyte names following
In the case of morphological observations, after collection, plants were dried and analysed in a laboratory using a stereomicroscope SMZ 800 (Nikon, Tokyo, Japan). The morphological characteristics of the investigated species were described (Table
Character / Feature | C. baltica | C. hispida | C. polyacantha | C. rudis |
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Plant axis | robust, slender | robust, thick | erect, robust | robust, thick |
Plant size | medium size, 6–27 cm high, 1–2 mm in diameter | medium large to large species, up to 18–70 cm high, 4–5 in diameter | medium large to large, 30–75 cm high, up to 5 mm in diameter | medium large to large plants, 23–65 cm high, up to 4–6 mm in diameter |
Color | light to dark green | green to greyish green | green to dark green | green to greyish green |
Incrustation | unincrusted | moderately to heavily incrusted | moderately incrusted | moderately to heavily incrusted |
Internodes | longer or as long as branches | longer than branches | similar length or longer (up to 2 times) than branches | up to 2 times longer than branches |
Branchlet | up to 8 branches in a whorl, stout to slender with 5–8 segments | 7–10 branches in a whorl, straight and rigid, with 5–9 segments | 8–10 branches in a whorl with 6–9 segments | 7–10 branches in a whorl, with 6–7 segments |
Cortification | diplostichous and slightly thylacanthous | diplostichous, aulacanthous, often isostichous on older internodes | diplostichous and tylacanthous occasionally irregular | diplostichous, strongly heterostichous and aulacanthous |
Spine cells | shorter than axis diameter, solitary or in pairs | solitary or in fascicles as long as the axis diameter | in bunches, as long or longer than the axis diameter | sparse in pairs, similar in length as plant axis |
Stipulodes | stipulodes in two rows similar in length to spine cells | stipulodes in two rows, uppers are similar to lowers | stipulodes in two rows, as long as axis diameter | stipulodes are in two rows, uppers similar to lowers |
Reproduction | monoecious | monoecious | monoecious | monoecious |
Oogonia | 540–1165 μm long, 515–650 μm wide | 415–1200 μm long, 520–770 μm wide | 625–1140 μm long, 450–615 μm | 890–1210 μm long, 415–750 μm wide |
Antheridia | 420–630 μmin diameter | 490–730 μm in diameter | 375–530 μm in diameter | 370–480 μm in diameter |
Oospores | black, 465–925 μm long and 335–645 μm wide | reddish brown to dark brown, 545–810 μm long, 390–760 μm wide | brown, dark brown or black, 485–900 μm long, 270–585 μm wide | brown to dark brown–almost black, 620–925 μm long, 395–835 μm wide |
Morphological features used in the analysis of features in four species in Chara section Hartmania. See Figure
Feature (see Figure |
Abbreviation |
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Number of branches in second branchlet whorl [n] | IL2 |
Mean length of branches in second branchlet whorl [cm] | SDL2 |
Mean number of corticated internodes on branchlets in the second whorl [n] | IOC2 |
Mean length of stipulodes at the second node [cm] | SDP2 |
Diameter of the internode above second branchlet whorl [cm] | SN2 |
Length of spine cells above second branchlet whorl [cm] | DO2 |
Number of branchlets in third branchlet whorl [n] | IL3 |
Mean length of branchlets in third branchlet whorl [cm] | SDL3 |
Mean number of corticated internodes on branchlets in third whorl [n] | IOC3 |
Mean length of stipulodes at the third node [cm] | SDP3 |
Diameter of the internode above third branchlet whorl [cm] | SN3 |
Length of spine cells above third branchlet whorl [cm] | DO3 |
In addition to the morphological observations, a molecular technique, sequencing of the plastid psaB gene, has been conducted. Total genomic DNA was isolated from fresh tissue using liquid nitrogen and a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Cells were disrupted using the Mixer Mill MM400 (Retsch, Haan, Germany). The quality and quantity of the DNA was determined on 1% TBE–agarose gel. The PCR amplification and sequencing of the psaB gene was accomplished using the primers described by
Prior to the phylogenetic analyses, the psaB DNA sequences were aligned using CLUSTAL W (
The specimens examined in the present study are described in detail in Table
The first three components in the PCA explained 25.1%, 21.3% and 17.5% of the total morphological variation. Four groups that correspond to the four species can be distinguished along the first and third axes (Fig.
In the discriminant analysis, specimens were assigned to species groups on the basis of the classical taxonomic approach. After analysis, the first three canonical functions accounted for 96% of the total variation (first 46%, second 24% and third 24%). The analysis showed that 11 out of the 14 characters were useful for differentiating the specimens. The other characters were not significant. The individuals of C. polyacantha, C. baltica and C. rudis form well-separated groups, and C. hispida overlaps C. rudis and C. baltica (Fig.
Analysis of the psaB gene of Chara species showed a smaller resolution than on the tree produced with sequences from the Nitella genus (
The results of numerous studies indicate that a combination of various morphological data with molecular sequences can be helpful for distinguishing charophyte species, as well as making various taxonomic decisions or explaining the phylogenetic relationship between species (
In comparison to other authors, and especially to more recently published data on the morphological features of charophytes (
The multivariate analysis of C. hispida and C. rudis based on vegetative traits gives some additional explanation of the taxonomy of species belonging to section Hartmania. The result obtained during the DCA seems to be clearer than obtained from the PCA, probably due to the different algorithms used in the analyses that can explain the differences: DCA emphasises characters that distinguish groups while suppressing the variation within groups, whereas PCA tends to accentuate the within-group variation (
The PCA results were used to demonstrate the differences among the species, and most specimens could be allocated to particular taxa. Both figures support the amalgamation of C. hispida and C. rudis, and maintenance of the species C. baltica and C. polyacantha as similar to how they were presented previously (
On the other hand, it supports the thesis that all these species are morphologically very similar, and that ‘transitional forms’ commonly exist between them. The so-called ‘transitional forms’ are probably not real hybrids, but rather forms that visualize possible plasticity that can be noted in the genus Chara. In this group of species: C. hispida, C. rudis, C. polyacantha and C. baltica ‘transitional forms’ are those that display features intermediate between species, or the features are not clear enough for determination. In the case of C. rudis and C. hispida, spine cells are the main distinguishing character, and they are normally in twos or threes in C. hispida, but in pairs lying one above the other along the axis in C. rudis (
The analysis of phylogenetic sequence data reveals a strictly close relationship between C. baltica, C. hispida, C. polyacantha and C. rudis (Fig.
Although morphological and molecular data separately are not ideal tools for species delimitation, together they are important and useful when combined with other types of data (
We have shown that morphological features allow for differentiation of the investigated Chara species. C. polyacantha formed separate clusters in both PCA and DCA, and C. rudis had intermediate features. Molecular analyses showed that all species definitely comprise one closely related group and no differentiation in the psaB variability between them has been found.