Journal of Systematics and Evolution 48 (4): 265–270 (2010) doi: 10.1111/j.1759-6831.2010.00089.x

Using DNA-based techniques to identify hybrids among linear-leaved Potamogeton plants collected in China

∗ ∗ 1,2Zhi-Yuan DU 1,2Chun-Feng YANG 1,2Jin-Ming CHEN ,† 3You-Hao GUO ,† 4Akeem Babalola KADIRI 1(Key Laboratory of Aquatic Botany and Watershed Ecology, Chinese Academy of Sciences, Wuhan 430074, China) 2(Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China) 3(Laboratory of Plant Systematics and Evolutionary Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China) 4(Department of Botany and Microbiology, University of Lagos, Lagos 100213, Nigeria)

Abstract It is well known that interspecific hybrids occur in the genus Potamogeton. The linear-leaved Potamogeton species commonly have highly variable morphological characteristics. Their hybrids often show similar vegetative characters to their parental species and their identification based solely on morphology is not always conclusive. In order to clarify whether there are any hybrids from the linear-leaved Potamogeton plants collected in China, we used internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequences to identify the hybrids. Using ITS sequence additivity, we identified four hybrids, namely P.orientalis (P.pusillus × P.oxyphyllus), P. pusillus × P. berchtoldii, P. foliosus × P. octandrus, and P. cristatus × P. octandrus. The latter three hybrids should be considered as new hybrids in Potamogeton. The maternal parents of the four hybrids were confirmed using chloroplast rbcL gene sequences. Key words hybridization, internal transcribed spacer, Potamogeton, rbcL.

The pondweeds Potamogeton L. (Potamoget- species) in Potamogeton (excluding Stuckenia;Guo& onaceae) are one of the most important plant genera Li, 1992; Wiegleb & Kaplan, 1998). Because of the in the aquatic environment (Haynes, 1975). As defined relatively frequent hybridization in Potamogeton, tradi- traditionally, the genus consists of two subgenera, Pota- tional morphologically based taxonomies are compli- mogeton and Coleogeton (Reichb.) Raunk. (Wiegleb & cated. Until now, only a few Potamogeton hybrids have Kaplan, 1998). Based on recent taxonomic treatment been identified in China (e.g. Wang et al., 2007; Zhang of Potamogeton, the subgenera Coleogeton has been el- et al., 2008; Du et al., 2009). evated to the generic level, giving it the correct name Over the past 5 years, field, herbarium, and labora- Stuckenia Borner (Lindqvist et al., 2006; Kaplan, 2008). tory studies have been conducted to assess Potamogeton Within the current Potamogeton, two morphological lin- populations in China. As part of these studies, we col- eages have been recognized, broad-leaved species and lected linear-leaved Potamogeton specimens from the linear-leaved species (Iida et al., 2004; Lindqvist et al., northeast, northwest, central, and southwest regions of 2006; Zhang et al., 2008). China. According to the species description of Guo & Potamogeton is a genus well known for the occur- Li (1992), Preston (1995), Wiegleb & Kaplan (1998), rence of interspecific hybrids (Kaplan & Fehrer, 2007). and Kaplan & Stepanek (2003), close inspection of the Wiegleb & Kaplan (1998) reviewed the taxonomic treat- specimens collected indicated that there were eight con- ment of Potamogeton and Stuckenia worldwide and firmed species. However, the linear-leaved Potamogeton assessed the validity of all taxa in these genera. In species commonly have highly variable morphological Potamogeton, hybrids are more likely to occur between characteristics, and their hybrids often show similar veg- species within a lineage than between species from dif- etative characters of their parental species. Thus, iden- ferent lineages (Fant et al., 2003). In China, there are tification of these hybrids on the basis of their mor- approximately 22 species (including eight linear-leaved phology is extremely difficult. Because of the limited number of studies on the identification of linear-leaved Potamogeton hybrids in China, it is not clear whether some hybrids collected during our field investigations Received: 28 December 2009 Accepted: 6 April 2010 have been misidentified as pure species. † These two authors have contributed equally to this study. ∗ Author for correspondence. E-mail: [email protected]; Tel.: +86-27- Because of the immense range of phenotypic plas- 68754775; Fax: +86-27-68752560. ticity of Potamogeton taxa (Kaplan, 2002), some authors

C 2010 Institute of Botany, Chinese Academy of Sciences 266 Journal of Systematics and Evolution Vol. 48 No. 4 2010 are skeptical about identifying Potamogeton hybrids manchuriensis A. Bennett) have been recorded in China morphologically and have stressed the need for more (Guo & Li, 1992; Wiegleb & Kaplan, 1998). However, convincing evidence (Les & Philbrick, 1993). In recent we did not find any plants of P. compressus and P. years, molecular techniques have been used to investi- manchuriensis in the areas we investigated in our re- gate the nature of some putative Potamogeton hybrids cent field study. Potamogeton berchtoldii Fieber and P. (Du et al., 2009). Nuclear internal transcribed spacer foliosus Rafinesque are morphologically very similar to (ITS) sequences of hybrids often show superimposed P.pusillus. They have not been recorded in China previ- nucleotides (additive patterns) of both parental species, ously. In our investigations, we collected P. berchtoldii so this region has been widely used to identify Pota- in the northeast and P.foliosus from many sites. In total, mogeton hybrids (Whittall et al., 2000, 2004; Kaplan & eight linear-leaved Potamogeton species were collected Fehrer, 2007). Maternal inheritance of chloroplast (cp) from eight provinces in the northeast, northwest, mid- DNA in the genus Potamogeton was confirmed in ex- dle, and southwest regions of China during 2005 and perimental hybrids (Kaplan & Fehrer, 2006). Thus, the 2008. The localities, taxa, number of samples included chloroplast sequences have been widely used to iden- in the molecular analyses, and GenBank accession num- tify the maternal parent of the hybrids (Fant et al., 2003; bers of the sequences are given in Table 1. In addition, Kaplan & Fehrer, 2007; Iida et al., 2007). In the present Table 1 lists the hybrids that have been confirmed by study, we used nuclear ITS sequences and chloroplast molecular techniques. About 5 g fresh leaves was har- rbcL gene sequences to test whether there are any hy- vested from each plant and dried in a zip lock plastic brids among the linear-leaved Potamogeton plants col- bag containing approximately 80 g silica gel. The sam- lected in China. ples were stored at room temperature until DNA was isolated in the laboratory.

1 Material and methods 1.2 DNA extraction, polymerase chain reaction am- plification, and sequencing 1.1 Plant material Total genomic DNA was extracted from dried A total of eight linear-leaved Potamogeton species leaf tissue using the cetyltrimethylammonium bromide (P.pusillus L., P.oxyphyllus Miquel, P.octandrus Poiret, (CTAB) protocol described by Doyle & Doyle (1990). P. cristatus Regel & Maack, P. acutifolius L., P. ob- The nuclear ITS region sequences were amplified us- tusifolius Mertens & Koch, P. compressus L., and P. ing primers ITS1 and ITS4 (White et al., 1990). The

Table 1 Localities, taxa, the number of samples, GenBank accession numbers of sequences, and voucher information Locality Taxon No. ITS rbcL Voucher samples no. Yilan, Heilongjiang Potamogeton berchtoldii Fieber 5 FJ956797 FJ956852 WH0121 Xiaoxingkai Lake, Heilongjiang P.acutifolius L. 3 FJ956800 WH0122 P.obtusifolius Mertens & Koch 3 FJ956799 FJ956854 WH0123 Mishan, Heilongjiang P.octandrus Poiret 3 FJ956785 FJ956840 WH0124 P.foliosus Rafinesque 2 FJ956795 FJ956850 WH0129 Raohe, Heilongjiang P.cristatus Regel & Maack 3 FJ956784 FJ956839 WH0132 Taibai, Shanxi P.pusillus L. 8 FJ956791 FJ956846 WH0135 Yichang, Hubei P.pusillus × P.oxyphyllus 25 FJ956901 & FJ956902 FJ968698 WH0143 Quanken, Congyang, Hubei P.octandrus Poiret 4 FJ956786 FJ956841 WH0125 P.cristatus Regel & Maack 6 FJ956782 FJ956837 WH0133 P.foliosus × P.octandrus 3 FJ956893 & FJ956894 FJ968694 WH0137 P.cristatus × P.octandrus 28 FJ956889 & FJ956890 FJ968692 WH0141 Guikou, Congyang, Hubei P.oxyphyllus Miquel 6 FJ956798 FJ956853 WH0136 P.octandrus Poiret 10 FJ956787 FJ956842 WH0126 P.foliosus × P.octandrus 17 FJ956895 & FJ956896 FJ968695 WH0138 Wuxing, Congyang, Hubei P.octandrus Poiret 5 FJ956788 FJ956843 WH0127 P.cristatus Regel & Maack 1 FJ956783 FJ956838 WH0134 P.foliosus × P.octandrus 13 FJ956897 & FJ956868 FJ968696 WH0139 P.cristatus × P.octandrus 21 FJ956891 & FJ956892 FJ968693 WH0142 Heqing, Yunnan P.foliosus Rafinesque 6 FJ956792 FJ956847 WH0130 P.octandrus Poiret 9 FJ956789 FJ956844 WH0128 P.foliosus × P.octandrus 6 FJ956899 & FJ956900 FJ968697 WH0140 Lijiang, Yunnan P.pusillus × P.berchtoldii 5 FJ956903 & FJ956904 FJ968699 WH0144 Linbao, Henan P.foliosus Rafinesque 6 FJ956793 FJ956848 WH0131 ITS, internal transcribed spacer.

C 2010 Institute of Botany, Chinese Academy of Sciences DU et al.: Identification of hybrid linear-leaved Potamogeton 267 chloroplast rbcL gene was amplified using primers 26 where there was a shift in the direct sequencing reading and 1375 described by Iida et al. (2007). The poly- frame between the two ITS types (Whittal et al., 2000, merase chain reaction (PCR) amplification was per- 2004; Kaplan & Fehrer, 2007). For each hybrid pop- formed in 25 μL reaction mixture containing 20 ng ulation, the purified ITS PCR products of two hybrid DNA, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, samples were cloned into the pUC19 vector (TaKaRa) 1.5 mmol/L MgCl2, 200 μmol/L each dNTP,0.4 μmol/L according to the manufacturer’s instructions and, for each primer, and1UTaqDNApolymerase(TaKaRa, each of them, four positive clones were sequenced. Dalian, China). The PCR amplification conditions for the ITS re- gion were as follows: initial predenaturation at 94◦Cfor 2 Results 5 min, followed by 30 cycles of 30 s at 94◦C, 30 s at 55◦C, and 1 min at 72◦C, with a final extension step of 2.1 Nuclear ITS region genotypes 10 min at 7◦C. The PCR amplification conditions for The boundaries of the ITS region were determined the chloroplast rbcL gene were the same as for ITS, by comparing previously published sequences (Wang except that the extension time was 2 min. Amplification et al., 2007). The ITS region (including 5.8S rDNA) of of genomic DNA was performed in a PTC-100TM ther- P.oxyphyllus, P.berchtoldii, and P.cristatus was 646 bp mocycler (MJ Research, Waltham, MA, USA). Amplifi- in length. The length of the ITS region of P. pusillus cation products were resolved on 1.5% agarose gels run was 647 bp and the length of P. foliosus and P. octan- at80Vin0.5× TBE (44.5 mmol/L Tris, 44.5 mmol/L drus was 631 bp, having a 15 bp deletion in ITS2 re- boric acid, and 1 mmol/L EDTA, pH 8.3), visualized gion. No intra- or interpopulation variation was detected by ethidium bromide staining, and photographed under within each pure species. For each population, the se- ultraviolet light. The size of amplification products was quence of only one sample of each species was sent to estimated using a 200 bp DNA ladder. GenBank (Table 1). Nine variable sites distinguished The PCR amplification products of both the ITS the P. foliosus sequences from those of P. octandrus region and rbcL gene were purified using a PCR prod- (Table 2). The sequences of P.cristatus and P.octandrus uct purification kit (SBS Genetech, Shanghai, China). had 16 different sites and a 15 bp deletion (Table 2). Six All the samples in Table 1 were sequenced directly substitutions and one indel distinguished the P.pusillus in both directions using the amplification primers. Se- sequences from those of P. berchtoldii (Table 3). The quencing was performed using the ABI Prism BigDye sequences of P. oxyphyllus and P. pusillus differed by terminator cycle sequencing ready reaction kit (Ap- 10 substitutions and one indel (Table 3). Hybrids have plied Biosystems, Foster City, CA, USA) and performed superimposed nucleotides of both parental species from on an ABI 3730 sequencer (Applied Biosystems). Se- chromatograms of direct sequences. No intra- or inter- quences were assembled using the program SeqMan population variation was identified within each hybrid 5.01 (DNASTAR, Madison, WI, USA) and aligned us- taxa. For each hybrid population, all samples showed the ing Clustal X (Thompson et al., 1997). The alignments same additive pattern in direct sequencing, and one hy- were then adjusted manually using the program EditSeq brid sample was cloned and sequenced. Each of the hy- 5.01 (DNASTAR). brids had five cloned sequences belonging to two types For hybrids, ITS polymorphisms at variable sites of ITS sequences corresponding to two pure species. that differentiated the parental species were identified as The Type 1 and Type 2 ITS sequences of the hybrids superimposed nucleotides (additive patterns) from chro- in Tables 2 and 3 were based on the cloned sequences. matograms of direct sequences; indel polymorphisms This phenomenon suggested that the two pure species in the ITS region were determined according to the site should be the parental species of the hybrids. The four

Table 2 Variable nucleotide sites in internal transcribed spacer (ITS) sequences for Potamogeton foliosus, P.octandrus, P.cristatus, and their putative hybrids (P.octandrus nucleotides are bolded) Taxon Variable nucleotide sites 021 051 102 109 113 198 199 211 217 225 411 424 432 477 512 563 565–579 604 633 635 P.foliosus (651bp) T T A A A T A T A T T A C C T C — — T A T P.octandrus (651 bp) ATGGATAGATTATTCT —— TA A P.cristatus (666 bp) T A A A C C G G G C C G T T T C 15 bp C G T P.foliosus × P.octandrus (Type 1) ATGGATAGATTATTCT —— TA A P.foliosus × P.octandrus (Type 2) T T A A A T A T A T T A C C T C — — T A T P.cristatus × P.octandrus (Type 1) ATGGATAGATTATTCT —— TA A P.cristatus × P.octandrus (Type 2) T A A A C C G G G C C G T T T C 15 bp C G T

C 2010 Institute of Botany, Chinese Academy of Sciences 268 Journal of Systematics and Evolution Vol. 48 No. 4 2010

Table 3 Variable nucleotide sites in internal transcribed spacer (ITS) sequences for Potamogeton berchtoldii, P. oxyphyllus, P. pusillus, and their putative hybrids (P.pusillus nucleotides are bolded) Taxon Variable nucleotide sites 025 051 198 222 442 451 491 514 548 557 558 565 626 P.oxyphyllus (666 bp) T A T – G T A T G C T T T P.berchtoldii (666 bp) T T C – G G C A G T C C T P.pusillus (667 bp) CTTATTCATTCCC P.pusillus × P.berchtoldii (type 1) CTTATTCATTCCC P.pusillus × P.berchtoldii (type 2) T T C – G G C A G T C C T P.pusillus × P.oxyphyllus (type 1) CTTATTCATTCCC P.pusillus × P.oxyphyllus (type 2) T A T – G T A T G C T T T hybrids were P. orientalis (P.pusillus × P.oxyphyllus), ents of these hybrids. Among the four confirmed hy- P. pusillus × P. berchtoldii, P. foliosus × P. octandrus, brids, the hybrid origin of P. orientalis (P. pusillus × P. and P.cristatus × P.octandrus. oxyphyllus) was reported by Wiegleb & Kaplan (1998). The other three hybrids, namely P. pusillus × P. berch- 2.2 Chloroplast rbcL haplotypes toldii, P. foliosus × P. octandrus, and P. cristatus × An 890 bp region of the rbcL gene sequence was P. octandrus, would be considered as new hybrids in obtained. The variable nucleotide sites in the rbcL se- Potamogeton. quences for P. berchtoldii, P. oxyphyllus, P. pusillus, According to the description of Potamogeton P. foliosus, P. octandrus, and P. cristatus are given in species by Guo & Li (1992), Preston (1995), Wiegleb Table 4. No intra- or interpopulation variation was iden- & Kaplan (1998), and Kaplan & Stepanek (2003), we tified within each taxa. For each population, the se- could distinguish eight pure species in our collections quence of only one sample of each species was sent to based on morphological characteristics. The leaves of GenBank (Table 1). Two confirmed hybrids, namely P. P.obtusifolius are obtuse or rounded at the apex and are orientalis (P.pusillus × P.oxyphyllus) and P.pusillus × easy to identify. Potamogeton octandrus and P.cristatus P. berchtoldii, had the same rbcL sequence as P. pusil- are the only species with floating leaves. Potamogeton lus. The hybrid P. foliosus × P. octandrus had an rbcL cristatus has an east Asian distribution and the dorsal sequence identical to that of P.foliosus, and the hybrid P. keels of its fruits are strongly crested with hooked ap- cristatus × P.octandrus had an rbcL sequence identical pendages. Potamogeton octandrus does not have cristate to that of P.cristatus. Reciprocal hybridization was not dorsal keels. Potamogeton pusillus, P. berchtoldii, and detected in these four hybrids. P. foliosus are morphologically very similar. The fruits of P. foliosus have distinct undulate and dentate dor- sal keels, whereas the dorsal keels of P. pusillus and 3 Discussion P. berchtoldii are indistinct. The stipules of P. pusillus are closed and tubular, whereas the stipules of P.berch- Using nuclear ITS sequence additivity, we con- toldii are open and convolute. The leaves of P. o x y - firmed that some linear-leaved Potamogeton plants col- phyllus and P. acutifolius are broader and longer than lected from China were hybrids. Using the chloroplast the above six species. Potamogeton oxyphyllus has an rbcL gene sequences, we identified the maternal par- east and southeast Asian distribution. Its stem is richly

Table 4 Variable nucleotide sites in rbcL sequences for Potamogeton berchtoldii, P. oxyphyllus, P. pusillus, P. foliosus, P. octandrus, P. cristatus,and their hybrids Taxon Variable nucleotide sites 085 129 281 388 510 849 P.berchtoldii TCGTTG P.oxyphyllus TCGTCG P.pusillus TCCTTG P.foliosus TCCTTG P.octandrus TCGTTG P.cristatus CTGGTA P.pusillus × P.oxyphyllus TCCTTG P.pusillus × P.berchtoldii TCCTTG P.foliosus × P.octandrus TCCTTG P.cristatus × P.octandrus CTGGTA

C 2010 Institute of Botany, Chinese Academy of Sciences DU et al.: Identification of hybrid linear-leaved Potamogeton 269 branched, but not compressed. Potamogeton acutifolius References has a strongly compressed stem and its flower has only Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh one carpel. Wang (1984) reported a new linear-leaved tissue. Focus 12: 13–15. Potamogeton species, P. chongyangensis W. X. Wang, Du ZY, Yang CF, Chen JM, Guo YH. 2009. Nuclear and chloro- from Chongyang, Hubei Province. However, in the re- plast DNA sequences data support the origin of Potamogeton cent classification of Potamogeton, Wiegleb & Kaplan intortusifolius J.B. He in China as a hybrid between P. p e r - (1998) regarded P. chongyangensis as a synonym of P. foliatus Linn. and P. wrightii Morong. Aquatic Botany 91: oxyphyllus, because this species has typical morpholog- 47–50. ical features of P.oxyphyllus. Fant JB, Kamau E, Preston CD. 2003. 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